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Poster 033 Follicle homing antigen presenting cells modulate

TH2 bias

Citation for published version: Bradford, B, Donaldson, D, Else, K & Mabbott, N 2014, 'Poster 033 Follicle homing antigen presenting cells modulate TH2 bias: Conditional knockout of CXCR5 on CD11c+ cells prevents protective TH2 response following T. muris infection' 9th European-Mucosal-Immunology-Group Meeting, Glasgow, United Kingdom, 1/10/14 - 12/10/14, . DOI: 10.13140/2.1.2667.2644

Digital Object Identifier (DOI): 10.13140/2.1.2667.2644

Link: Link to publication record in Edinburgh Research Explorer

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Download date: 05. Apr. 2019 IMMUNOLOGY ABSTRACTS

Poster Abstracts

001 002 Cryptosporidium parvum subverts the host innate immune Faecalibacterium prausnitzii strain HTF-F and its extracellular response through manipulation of CRAMP expression during polymeric matrix attenuate clinical parameters in DSS-induced neonatal infection colitis W. Guesdon1, T. Pezier1, F. Drouet1, S. Menard2, F. Laurent1 & O. Rossi1, M. Schwarzer2, T. Hudcovic2, D. Srutkova2, S. Duncan3, S. Lacroix-Lamande1 H. Flint3, J. Samsom4, H. Harmsen5 & J. Wells1 1Animal Health, UMR1282 Inra-Universite, Nouzilly, France, 1Host-Microbe Interactomics, Wageningen University, Wageningen, 2Toxalim, UMR 1331 INRA/INP/UPS, Toulouse, France Netherlands, 2Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3Rowett Institute of Nutrition and Health, University Due to the immaturity of their immune system, neonates are highly Aberdeen, Aberdeen, UK, 4Department of Pediatrics, Erasmus Medical sensitive to intestinal infections. During the neonatal period, antimi- Center, Sophia Children’s Hospital, Rotterdam, Netherlands, crobial peptide (AMP) expression differs substantially from that of 5Department of Medical Microbiology, UMCG, Groningen, Netherlands adults as this is the case for the cathelicidin-related antimicrobial peptide CRAMP expressed preferentially in the neonatal period while Introduction: A decrease in the abundance and biodiversity of intes- conversely other AMPs such as Reg3c are expressed later in life. tinal bacteria within the Firmicutes phylum has been associated with Among enteric neonatal diseases, Cryptosporidiosis is a zoonotic inflammatory bowel disease (IBD). In particular, the anti-inflamma- disease and is highly prevalent in in young infant less than 5 years tory bacterium Faecalibacterium prausnitzii, member of the Firmi- old in underdeveloped country and in neonatal ruminants world- cutes phylum and one of the most abundant species in healthy wide. Cryptosporidium parvum is the etiological agent of this diar- human colon, is underrepresented in the microbiota of IBD patients. rheal disease and infects exclusively epithelial cells. Innate immunity Method: In this study we investigated the capacity of F. prausnitzii is important to control the acute phase of infection in neonates with strain A2-165, the biofilm forming strain HTF-F and the extracellular dendritic cells and IFNc playing a major role. Antimicrobial peptides polymeric matrix (EPM) isolated from strain HTF-F, to suppress inflam- are important contributors of innate immunity, but the role of mation in the mouse dextran sodium sulphate (DSS) colitis model. CRAMP, which is elevated in the intestine of neonates has never Results: The two F. prausnitzii strains have anti-inflammatory effects been investigated during Cryptosporidiosis so far. in the DSS colitis model.F. prausnitzii HTF-F is more effective than In this work, we used the neonatal murine model of cryptosporid- A2-165 partly because of the immune-regulating properties of the iosis and unlike all the other antimicrobial peptides analyzed EPM. The immunomodulatory effects of the EPM are mediated CRAMP expression in the intestinal epithelial cells was significantly through the TLR2-dependent modulation of IL-12 and IL-10 cyto- reduced during infection. This reduced CRAMP expression is inde- kine production in antigen presenting cells. Both F. prausnitzii HTF- pendent of IFNg, a cytokine strongly produced during infection but F and the EPM may have a therapeutic use in IBD. also Myd88 and gut flora independent. When C. parvum infected neonatal mice orally received exogenous CRAMP to compensate the reduced expression of this AMP, the parasitic load of neonates was 003 significantly decreased. In addition, when free parasites were in Roseburia hominis regulates immunity through TLR-5 dependent direct contact with CRAMP, this AMP affects the viability of spor- responses ozoites, the first free infectious form of this parasite. All together, these data suggest that C. parvum induces the reduction of CRAMP A.M. Patterson, E. Monnais, A.G.P. Coutts, N.H. McKenzie, expression to escape the anti-parasiticidal effect of CRAMP. The E. Logan, M. Delday, I.E. Mulder & G. Grant molecular mechanism by which the parasite subverts epithelial- Gut Immunology, Life Sciences Innovation Building, Cornhill Road, derived CRAMP production is currently under investigation. Aberdeen, UK Commensal bacteria play a fundamental role in maintaining immune homeostasis a complex balance between immune stimulation and appropriate responsiveness and immune suppression and tolerance. Break- down of these immune regulatory processes lead to chronic inflammation associated with intestinal dysbiosis. The immune modulat- ing properties of Roseburia hominis, a flagellate gut anaerobe, were investigated in colonised conventional and colitic mouse models. Oral administration of Roseburia hominis to conventional mice with colitis caused by Dextran Sodium Sulphate altered intestinal immune responses, attenuated gut inflammation and reduced the severity of colitis. This protection was associated with a modulation and expansion of the T regulatory cell population triggered by R. hominis. The role of flagellin in directing these immune responses was investigated in vitro and in vivo. Part funded by RESAS, Scotland and part funded by GT Biologics.

004 Stimulation of adenotonsillar MNC by CpG-DNA significantly A specific tolerance inducing vector for therapeutic treatment of increased TFH number and that was correlated with HA-specific autoimmune diseases antibody production following influenza antigen stimulation. Co-incubation with purified plasmacytoid dendritic cells significantly C. Hansson, K. Schon€ & N. Lycke enhanced the CpG-DNA-mediated antibody production. Microbiology and Immunology, Gothenburg University, Gothenburg, Conclusion: A prominent number of TFH were shown in human Sweden NALT especially in children and that were highly active in B cell- Introduction: We have developed a novel platform for intranasal treat- help for antibody production. CpG-DNA promoted NALT TFH cells ment of autoimmune diseases in which ADP-ribosylation determines which correlated with the enhancement of HA-specific antibody pro- whether immunity or tolerance is induced. Hence, cholera toxin duction. To enhance vaccine immunogenicity by promoting TFH A1-subunit based immunomodulation through the CTA1-peptide-DD function may be an effective vaccination strategy. fusion protein promotes enhancement, while inactive mutants induce suppression. Whereas the target population after intranasal administration of both constructs was CD103+CD11blow DC’s, both active and inactive mutants induced strong CD4 T cell priming, but they differen- 006 tially affected CD4 T cell differentiation. This way inactive mutant con- A20/TNFAIP3 as a brake on intestinal inflammation and structs generated regulatory CD4 T-cells producing IL-10 and effectively tumorigenesis preventing experimental autoimmune encephalitis (EAE) and collagen- L. Vereecke1, S.V. Silva2, T. Billiet3, J.H. van Es4, C. McGuire1, induced arthritis (CIA), when carrying relevant peptides. Targeted DCs K. Slowicka1, M. Sze1, M. van den Born4, G.D. Hertogh5, expressed low levels of CD80, CD86 and CD40, while, by contrast, H. Clevers4, J. Raes6, P. Rutgeerts3, S. Vermeire3, R. Beyaert1 & ADP-ribosylating CTA1-peptide-DD constructs gave significantly G. van Loo1 enhanced co-stimulation. Suppression was global in that regulatory 1Inflammation Research Centre, Technologiepark 927, Ghent, Belgium, T-cells following treatment were able to suppress adoptively transferred 2Department of Microbiology and Immunology, Rega In, Belgium, € naıve CD4 T cells and could subsequently maintain tolerance. 3Department of Clinical and Experimental Medicine, Leuven, Belgium, Conclusion: The use of specifically tolerance-inducing fusion pro- 4Hubrecht Institute for Developmental Biology and S, Utrecht, teins may be a way forward in the search of effective treatments Netherlands, 5Department of Morphology and Molecular Pathology, against autoimmune diseases. The immunomodulating effect on the 6 + Leuven, Belgium, Department of Microbiology and Immunology, Rega CD103 CD11blow DCs will be described and the requirement for In, Leuven, Belgium CTA1-binding to Gs alpha evaluated in detail, using Cre-lox Gs alpha-deficient mice. Intestinal immune homeostasis is established through complex interaction between commensal bacteria, the intestinal epithelium and mucosal immune cells. A defective interaction between these compartments can result in intestinal pathology. A crucial mediator in 005 establishing this homeostasis is the transcription factor NF-kappaB, A study on T follicular helper cells (TFH) in human NALT and since it regulates multiple protective mechanisms in intestinal epithe- effect of CpG-DNA on TFH -mediated antibody production lial cells (IEC’s), and pro-inflammatory responses in mucosal immune cells. NF-kappaB activation is tightly controlled by its nega- A. Aljurayyan1, N. Upile2, C. Vaughan2, C. Xie2, R. Sharma2, 3 3 4 1 tive feedback regulator A20, which in addition has pronounced anti- H. Beer , M.S. McCormick , S. Gordon & Q. Zhang apoptotic functions. Genetic studies identified polymorphisms in the 1Clinical infection, Microbiology and Immunology, Ronald Ross 2 A20 locus associated with multiple inflammatory and auto-immune Building, Liverpool, UK, ENT Department, Alder Hey Children’s pathologies, including coeliac and Crohn’s disease. To investigate the Hospital, Liverpool, UK, 3ENT Department, Royal Liverpool University 4 physiological role of A20 in intestinal immune homeostasis, we gen- Hospital, Liverpool, UK, Liverpool School of Tropical Medicine, erated tissue specific A20 knockout mice. We found that A20 has Liverpool, UK predominant anti-apoptotic functions in IEC’s and predominant Background: T Follicular helper cells (TFH) have been identified as anti-inflammatory functions in myeloid cells. By deleting A20 in a distinct CD4+ T cell subset and considered important for germinal both IEC’s and myeloid cells, we generated a novel mouse model of centre function and critical for T cell-dependent B cell antibody pro- intestinal inflammation which is characterized by early Paneth and duction. Therefore it may be an effective strategy to enhance vaccine goblet cell loss. Older mice suffer from ileitis and severe colitis which immunogenicity by promoting TFH numbers/function in humans. often progresses to colorectal cancer development. These findings Adenotonsillar tissues are nasopharynx-associated lymphoid tissue suggest that defects in proper A20 function may contribute to the (NALT) and important in response to intranasal vaccination. development and progression of inflammatory bowel disease and Aims: We analysed TFH numbers and function in NALT from children cancer in humans. and adults and studied whether and how CpG-DNA promotes TFH function leading to enhancement of mucosal immunity to influenza. Methods: Mononuclear cells (MNC) were isolated from adenotonsillar 007 tissue of children and adults, and TFH number (CD4+/CXCR5+/ ICOS+) and function were analysed by flowcytometry and intracellular Ablation of macrophages by prolonged blockade of CSF1R cytokine staining. Purified CXCR5+ TFH were co-cultured with B cells signalling depletes M-cell differentiation in the intestinal with/without influenza antigens and CpG-DNA. Haemagglutinin (HA)- epithelium specific antibody production was analysed by ELISA and Elispot assay. A. Sehgal1, D. Donaldson2, D. Hume2 & N. Mabbott2 Results: A prominent number of TFH were identified in NALT 1University of Edinburgh, Easter Bush, Midlothian, Edinburgh, UK, which were considerably higher than PBMC, and the mean NALT 2The Roslin Institute and RDSVS, University of Edinburgh, Easter TFH number in children was significantly higher than in adults. Bush, Midlothian, Edinburgh, UK NALT TFH were shown to express high levels of IL-21 and that were important for B cell antibody production. Co-culture of purified The gut-associated lymphoid tissue (GALT), including Peyer’s TFH but not non-TFH with B cells promoted antibody production. patches (PPs) and isolated lymphoid follicles lack afferent lymphatics

and directly sample mucosal antigens by specialized epithelial cells in 009 the follicular associated epithelia (FAE), known as microfold (M) Adjuvants modulate the immune response elicited by oral cells. M cell differentiation has been attributed to factors released by vaccination with adenovirus based vectors cells within the sub-epithlelium dome of PP but very little is known J. Revaud1, J. Shaw1, Y. Unterfinger1, J. Maye2, N. Versille2, about factors within crypts that may affect M cell differentiation. Here B. Klonjkowski1, J. Ben-Arous2 & J. Richardson1 we provide evidence of macrophages in close contact with Paneth 1UMR Virologie, ENVA, Maisons-Alfort, France, 2Seppic, France cells in intestinal crypts may play a role in M cell differentiation. In our study, mice were treated with an anti-CSF1R monoclonal Introduction: Oral delivery of vaccines is effective in inducing muco- antibody (M279) for 6 weeks. This led to a complete ablation of sal immunity and thus represents a highly relevant route for improved CD68+CD11c+Csf1r-EGFP¬ macrophages in the gut. Our study vaccines. At present, however, most orally delivered vaccines are live demonstrates a loss of M cells and a reduction in antigen uptake attenuated vaccines, which present a risk of reversion to virulence. in the FAE of PP of mice. We also found a loss of Paneth cells in Vaccines derived from recombinant adenoviruses (rAd) that have intestinal crypts however we found no expression of CSF1R on Pa- been rendered replication incompetent provide a safer alternative. neth cells indicating this may be due to loss of macrophages in Such vaccines elicit potent humoral and cellular immune responses close contact with intestinal crypts. These data suggest that macro- (IR) after parenteral delivery, but have proven less effective as oral phages may have a more intimate relationship with Paneth cells vaccines, potentially in relation to such factors as insufficient stability than previously understood and may control M cell differentiation of the vector in the gastro-intestinal environment, sub-optimal pene- within the FAE by providing a scaffold for intestinal crypt prolifer- tration of the intestinal barrier, insufficient presentation by antigen- ation function. presenting cells or a local immunological context biased towards immunological tolerance. It is possible that many of these obstacles could be overcome by appropriate use of adjuvants. Objective: The objective of this work was to determine the effect of 008 several classes of adjuvant on the amplitude and quality of the Activation and differentiation of human lamina propria (myeloid) immune response after oral administration of rAd. cells during the initiation of intestinal inflammation Method: To this end, a vector derived from a type 5 human adenovirus and encoding the reference antigen ovalbumin (ova) was J. Gras1, M. Paparella1, B. Brors2, T. Giese1, F. Lasitschka1, administered to mice by the intra-gastric route in the presence of G. Wabnitz1, M. AlSaeedi1, S. Schiessling1, S. Meuer1 & various adjuvants. The humoral IR against ova was evaluated in J. Schroder-Braunstein€ 1 serum, feces and vaginal lavage fluids by ELISA. For two adjuvants, 1University Hospital Heidelberg, Heidelberg, Germany, 2German the cellular IR against ova was assessed in spleen, intestine (ileum) Cancer Research Center, Heidelberg, Germany and mesenteric lymph nodes by intracellular cytokine staining and Mucosal lamina propria cells of IBD patients are highly activated in flow cytometry. contrast to the hyporesponsive phenotype of these cells under homeo- Results: The initial results indicate that the tested adjuvants can static conditions. Given, that human studies are based on patient biop- indeed modulate the humoral and cellular IR after oral administra- sies taken years after disease onset, the molecular events initially tion of rAd, but each in a different manner. It should thus be possi- causing this inflammation are still largely unknown. In this study, we ble to modify, both quantitatively and qualitatively, the IR elicited aimed to characterize early activation mechanisms of lamina propria by orally delivered rAd by using adjuvants. Further work will address cells including myeloid cells by utilizing an ex vivo human intestinal the impact of the different adjuvants at sequential steps involved in organ culture model which was recently described by us. In this model, induction of the IR. healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment which resulted in the activation of lamina propria immune cells and their emigration out of the tissue. Global gene expression analysis of lamina propria cells obtained by laser-capture 010 microdissection prior and after loss of the epithelial layer revealed an CCR2 expression defines a functionally distinct population of induction of myeloid (activation) surface receptors such as CD80, conventional DCs in intestinal mucosa CD83, CCR7, as well as inflammatory mediators like IL1B, IL-23p19 C. Scott1, C. Bain2, P. Wright2, D. Sichien1, M. Guilliams1, in lamina propria cells in situ. Upregulation of these markers/media- B. Lambrecht1, S. Milling2 & A.M. Mowat2 + + tors was confirmed in emigrated HLA-DR CD11c CD14- and HLA- 1Inflammation Research Centre, Ghent, Belgium, 2Institute of Infection, + + + DR CD11c CD14 lamina propria myeloid cells when compared to Immunity and Inflammation, University of Glasgow, Glasgow, UK peripheral blood myeloid cells by qRT-PCR and/ or flow cytometry. In addition, bioinformatic analysis of in situ gene expression profiles The origin and functions of mononuclear phagocytes (MPs) in the revealed the induction of signalling pathways not yet associated with intestine have been a source of confusion for some time, partly the activation of lamina propria (myeloid) cells under inflammatory because overlapping markers such as CD11c, CD11b, CX3CR1, conditions, e.g. the response to unfolded protein. The upregulation of MHC class II and CCR2 have been used to distinguish dendritic cells ^ (DCs) from macrophages (MFs). While CD103+ intestinal MPs known target genes of this pathway such as GADD153, C/EBPa, and – HSPA5 in the latter cell population was subsequently confirmed in situ appear to be genuine DCs, the nature of CD103 MPs remains con- by qRT-PCR and/ or immunofluorescence. These findings provide troversial. By combining phenotypic, gene profiling and kinetic – + insight into molecular events underlying the change from a hypore- analysis, we show that CD103 CD11b MPs in both the small and sponsive to a highly activated phenotype in lamina propria (myeloid) large intestine comprise distinct populations of DCs and MFs. + cells at the very onset of inflammation. CD64-CD103-CD11b MPs are classical DCs, being derived from Flt3L-dependent DC-committed precursors, migrating in afferent lymph, turning over rapidly in vivo and priming na€ıve CD4+ T cells. In contrast, the CD64+ subset of CD103-CD11b+ MPs is sessile and avidly phagocytic, derives from Ly6Chi monocytes in a non-Flt3- dependent manner and turns over slowly in vivo. Surprisingly however, the monocyte/MF lineage marker CCR2 is expressed by a

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 15 significant proportion of CD103-CD11b+ DCs and there is a selec- epithelium and causes profuse diarrhea that can be life threatening tive decrease in CD103-CD11b+ DCs in mice lacking this chemokine in very young children and immunocompromised individuals. Neo- receptor. CCR2+CD103- DCs in the LP express IRF4, their presence natal mice are highly susceptible to C. parvum but the infection is is partially dependent on this transcription factor in vivo and they self-limited, whereas adult mice are resistant unless immunocompro- have a selective ability to drive IL-17a production by T cells both in mised. Accumulating evidences obtained with the neonatal mouse vivo and in vitro. Furthermore, an equivalent population of model demonstrate the key role of innate immunity to control the CCR2+CD103- DCs is present in human intestine Together, these acute phase of the infection. Conditional depletion of CD11c+ cells data highlight the heterogeneity of intestinal MPs and reveal a bona demonstrated their essential role for the control of the infection both fide population of CCR2+ conventional DCs, which is involved in in neonates and adults (1). We therefore investigated the contribu- priming mucosal Th17 responses. tion of CD103+ DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in CD103+ DC in the intestinal lamina propria during the first weeks of life and artificially increasing the number of intestinal CD103+ DC by 011 administering FLT3-L significantly reduced susceptibility to the CCR7 expression of intestinal myeloid cells and their migratory infection. We next identified an unexpected mechanism of recruit- potential ment of the CD103+DC during the infection with several transgenic + G. Desalegn1, G.-L. Moschovakis1, A. Schridde1, L. Utrianen2, mouse models. Indeed, rapid recruitment of CD103 DC was S.W. Milling2,R.Forster€ 1 & O. Pabst1 depending on the production of CXCR3-binding chemokines pro- ɣ 1Institute of Immunology, Hannover Medical School, Hannover, duced by IEC in response to IFN . In addition to this key role in + ɣ Germany, 2Institute of Infection, Immunity and Inflammation, CD103 DC recruitment, IFN is known to inhibit intracellular para- University of Glasgow, Glasgow, Scotland site development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFNɣ in the lamina propria and the + + CD103 DCs and CX3CR1 lamina propria (LP) cells represent draining lymph nodes. Thus, CD103+DC are key players in the non-overlapping populations with distinct precursors in the small innate immune control of C. parvum infection in the intestinal epi- + intestine. Our group suggested that CD103 DCs but not CX3CR1hi thelium. The relative paucity of CD103+ DC in the neonatal intestine cells migrate to the mesenteric lymph nodes (mLN) where they contributes to the high susceptibility to intestinal infection. Our € prime naıve T cells. We observed that CX3CR1int cells are present work makes a substantial increase in the understanding of the role in intestinal lymph but not CX3CR1hi cells (Schulz O et al, 2009). of immune effectors involved in the control of the acute phase of C. In contract, recently published data indicated that in microbiota- parvum infection and paves the way to immune modulation strate- depleted mice CX3CR1hi cells become migratory and enter into lym- gies (2) targeting intestinal DC for strengthening neonatal immune phatics and mLN in a CCR7-dependent manner (Diehl GE et al, system against enteric infections. 2013). We therefore established a novel mouse model to monitor (1) Lantier et al. PLoS Pathog. 2013 CCR7 expression and its regulation in vivo. In this mouse model (2) Lantier et al. J Infect Dis. 2014 KW Dendritic Neonate Innate insertion of GFP disrupts the function of the CCR7 gene. Upon Infection stimulation, cells from CCR7-GFPki/ki mice up-regulate CCR7/GFP but are not able to migrate because of the disrupted CCR7. In contrast, cells from CCR7-GFPki/wt mice can up-regulate CCR7 and are capable of migrating to the draining LN. Mice were treated with 013 antibiotic or left untreated, and gavaged with R848 14 hours prior CD11b is required for oral tolerance by regulating Treg – to analysis. Whereas intestinal CD103+ and CD103 DCs up-regu- frequencies in the small intestine lated CCR7/GFP after R848 stimulation both in antibiotic treated A. Schridde1 & O. Pabst2 and untreated mice, CD64+ macrophages didn’t express CCR7 even 1Institute of Immunology, Carl-Neuberg-Str. 1, Hannover, Germany, after microbiota depletion. CD11c+MHCII+CD64-CD103- LP cells 2Institute of Molecular Medicine, Pauwelsstraße 30, Aachen, Germany expressed the chemokine receptor. Moreover, in CXCR1GFP/+ mice, CX3CR1int but not CX3CR1hi cells were found in the mLN after Intestinal tolerance requires the activation of FoxP3+ regulatory T R848 in the antibiotic treated and untreated mice. Thus, CD103+ cells (Tregs) in the mesenteric lymph nodes by CD103+ dendritic CX3CR1- and CD103-CXCR1int DCs express CCR7 upon stimula- cells and their subsequent homing to the intestine. Signalling by resi- tion. In contrast, intestinal macrophages characterized by a dent CD11b+F4/80+CX3CR1high macrophages present in the intesti- CD64+CX3CR1hi phenotype did not upregulate CCR7 irrespective nal lamina propria leads to further Treg expansion. We have of the microbiota status in these mice. previously shown that the high expression of CX3CR1 by resident macrophages is necessary for their expression of IL-10. In CX3CR1- deficient mice, reduced IL-10 expression leads to a loss in Treg expansion in the small intestinal lamina propria and hence abrogates 012 oral tolerance. CD103+DC play a key role in the innate immune mechanisms Here, we show that signalling via CD11b, in addition to CX3CR1, protecting the epithelium against neonatal enteric infections is also involved in regulating oral tolerance. Using delayed type F. Laurent, L. Lantier, S. Lacroix-Lamande, F. Drouet, hypersensitivity measurements, we observed that CD11b-deficient W. Guesdon & A. Gnahoui-David mice failed to establish oral tolerance. To investigate Treg induction AH, Centre Val de Loire UMR ISP bat 213, Nouzilly, France in the mLN and Treg frequencies in the small intestinal lamina propria in vivo, we performed adoptive transfer experiments in Neonates are generally more susceptible than adults to infectious dis- CD11b-/- mice. Like in CX3CR1-deficient mice, Treg cells were eases. Their intestinal immune system is in development and subject readily induced in the mesenteric lymph nodes at frequencies com- to numerous changes after birth, facing the colonization by the parable to wild type mice. Moreover, expression levels of the gut commensal flora, alimentary antigens, and aggression by enteric homing molecules a4b7 and CCR9 were unimpaired. On the con- pathogens. Cryptosporidium parvum is a highly prevalent zoonotic trary, Treg frequencies in the lamina propria were reduced in protozoan parasite that develops only in the gastrointestinal CD11b-/- mice, possibly explaining the oral tolerance defect.

In order to gain a comprehensive picture of the pathways that 015 might be changed in resident macrophages due to the deficiency of Characterisation of antigen-specific CD4+ T cells by using MHC CD11b and CX3CR1 we carried out complex transcriptome and class II tetramers cytokine analysis. Based on these studies, we intend to define a pro- G. Prota, E. Pettini, G. Pozzi, D. Medaglini & A. Ciabattini file of intestinal macrophages that is important for their tolerogenic L.A.M.M.B., Dipartimento di Biotecnologie Mediche, Universitadi function. These results in turn will open insights into pathways that Siena, Siena, Italy can be targeted for regulating Treg frequencies and oral tolerance establishment. In the context of vaccination, CD4+ T-cell primary activation can be considered as an early biomarker of vaccine immunogenicity since it is required for both the induction of high-affinity antibodies and immune memory. To this aim, we have here characterized the 014 Ag-specific CD4+ T cell response following immunization with a Changes in the pattern of expression of Fatty Acid Binding Mycobacterium tuberculosis vaccine antigen plus a liposome-based Proteins in small intestine in untreated Coeliac Disease adjuvant administered by two different routes (parenteral versus 1 2 2 3 4 mucosal). Ag85b-specific MHC class II tetramers were employed for N.B. Arias , M. Garcia , C. Bondar , L. Guzman , M. Yantorno , + B. Corsico5 & F. Chirdo2 detecting Ag-specific CD4 T cells. C57/BL6 mice were primed by 1 2 the subcutaneous or nasal route, and the clonal expansion of Ag-spe- LISIN-IIFP, INIBIOLP. UNLP, La Plata, Argentina, LISIN-IIFP. + UNLP, La Plata, Argentina, 3Servicio de Gastroenterologia. Hospital de cific CD4 T-cells was analyzed into respective draining lymph nodes Ninos.,~ La Plata, Argentina, 4Servicio de Gastroenterologia. Hospital (LNs) and spleens by using Ag85b280-294 MHC class II tetramers. San Martin, La Plata, Argentina, 5INIBIOLP. UNLP, La Plata, Cells were also characterized for the expression of surface markers Argentina and the production of cytokines. Prime-boost experiments, that combined the nasal and parenteral routes, were also performed and Coeliac disease (CD) is an immune-mediated enteropathy that devel- the cellular, as well as the humoral response were analysed. Tetra- ops in genetically susceptible individuals following exposure to die- mer-positive CD4+ T cells were detected into respective draining tary gluten. Fully differentiated epithelial cells express two isoforms LNs with a peak 7-9 days after priming (about 0.12 and 0.3% of of fatty acid binding proteins (FABPs): intestinal and liver, I- and total CD4+ T cells, following nasal and parenteral priming respec- LFABP, respectively. I- and LFABPs belong to a family of small cyto- tively), and declined at day 14. Priming by the SC route induced a solic proteins which bind and transport long chain fatty acids, but stronger CD4+ T cell clonal expansion respect to the nasal one, and also have other important biological roles in signalling pathways, a higher dissemination towards spleens and lungs. Upon parenteral particularly those related to PPARs which link lipid metabolism and immunization about 20% of Ag85b-specific primed CD4+ T cells inflammatory process. were T follicular helper (Tfh) cells (CXCR5+PD1+), and about a The aim of this work was to analyse the pattern of expression of 30% Th1 (CXCR3+). CD4+ T cells primed by the parenteral route I- and LFABP in small intestine in normal tissues and in those from were efficiently boosted by nasal as well as parenteral immunization. untreated CD patients as well as the evaluation of IFABP serum lev- Ag85b280-294-MHC class II complexes can be efficiently used for els in CD patients and non-CD controls. Expression of I- and studying the immunogenicity of a tuberculosis vaccine in the mouse LFABP in duodenal tissues was assessed by confocal fluorescence model, and characterizing the cellular response after both priming microscopy using specific polyclonal antibodies and by quantitative and boosting. Here, we showed that a single parenteral immuniza- PCR. Immunofluorescence analysis showed a differential pattern of tion with a subunit tuberculosis vaccine induced the expansion of expression for both FABPs when normal tissue and severe enteropa- Ag-specific CD4+ T cells that differentiated mainly into Tfh and thy were compared. I- and LFABP were expressed in the epithelium Th1, and that this vaccine formulation primes the immune system in healthy mucosa and in the remaining epithelium in partial or better by the parenteral than the nasal route. total villus atrophy. Slight labelling for IFABP was also observed in the crypts in healthy mucosa. Remarkably, FABPs expression was clearly increased in the crypts of intestinal mucosa in untreated CD. Quantitative PCR analysis showed that mRNA levels for LFABP were 016 higher than those for IFABP in normal tissue. Employing bactin as Characterization of gastric tissue-resident memory CD8+ T cells housekeeping gene, I- and LFABP mRNA levels were lower in duo- from children, adults and the elderly denal samples from adult untreated CD patients than in healthy con- J. Booth, F.R. Toapanta, R. Salerno-Goncalves, S. Patil, trols (P < 0.01). In paediatric population this difference was not H.A. Kader, A.M. Safta, S.J. Czinn, B.D. Greenwald & M.B. Sztein observed. On the other hand, when villin was used as reference, the Department of Pediatrics, Medicine and CVD, 685 West Baltimore levels of both FABPs were increased in enteropathy. Using a com- Street, Baltimore, USA mercial quantitative ELISA, we observed that IFABP serum levels were higher in adult untreated CD patients compared with healthy The main orchestrators of protective immunity in the stomach are T controls (P < 0.0001), patients on Gluten free diet (P = 0.023) and cells. However, limited information is available on the presence and inflammatory bowel disease patients (P < 0.001). function of the gastric T subsets mostly due to the difficulty in iso- In conclusion, IFABP, which is likely released from the damaged lating high numbers of viable cells from human gastric biopsies. To enterocyte, can be used as a biomarker providing additional infor- overcome this shortcoming, we optimized a cell isolation method mation in diagnosis and follow up of CD. The marked increase in I- that yielded high numbers of viable lamina propria mononuclear and LFABP expression in the crypts may reflect an accelerated rate cells (LPMC) from gastric biopsies obtained during upper GI endos- of enterocyte differentiation as compensatory mechanism due to the copy . Classic memory T (TM) subsets were identified in gastric increase in epithelial loss. LPMC obtained from children, adults and the elderly using flow cytometry. A dominant effector memory (TEM) phenotype was observed in gastric LPMC CD8+ T cells in all age groups. We then evaluated whether these cells represented a population of gastric tissue-resident memory T (TRM) cells by assessing expression of CD103 and CD69. The vast majority of gastric LPMC CD8+ T cells

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 17 either co-expressed CD103/CD69 (>70%) or expressed CD103 alone 018 (~20%). Thus, gastric LPMC CD8+ T cells had the characteristics of CTA1-3M2e-DD; a broadly protective vaccine candidate against TRM cells. In addition, gastric CD8+ TRM cells produced multiple influenza virus cytokines (IFN-c, IL-2, TNF-a, IL-17A, MIP-1b) and up-regulated D.G. Eliasson1, K. Schon1, X. Saelens2 & N. Lycke1 CD107a upon stimulation with mitogens. Furthermore, gastric CD8+ 1Microbiology and Immunology, Medicinaregatan 7A, Gothenburg, TRM demonstrated differences in the frequency, susceptibility to Sweden, 2VIB-Molecular Biomedical Research, University of Ghent, activation and cytokine/multi-cytokine production profiles among Belgium the age groups. Most notably, children’s gastric CD8+ TRM cells responded differently to stimuli than gastric CD8+ TRM cells from Few mucosal vaccines have successfully been launched on the market. adults or the elderly. In conclusion, we demonstrate the presence of This is partly because of the lack of potent mucosal vaccine adjuvants. gastric CD8+ TRM which exhibit diverse functional characteristics in We have generated a targeted fusion protein based on the non-toxic children, adults and the elderly. mucosal adjuvant CTA1-DD, harboring tandem repeats of the conserved matrix protein 2 ectodomain (M2e) of influenza A virus. This mucosal influenza vaccine candidate, CTA1-3M2e-DD, was found to confer strong protective immunity against a lethal challenge infection 017 with live influenza virus in mice. Although anti-M2e IgG antibodies Constitutive Type I Interferon, via STAT1 activation, selectively correlated with protection in Balb/C mice, this correlation was less promotes regulatory T cell function in the healthy human prominent in congenic Balb/B mice. These mice exhibited poor resis- intestine, but not in IBD tance to the challenge infection despite comparable anti-M2e IgG E. Giles1, T.J. Sanders1, N.E. McCarthy1, I. Sanderson2, antibody titers. Hence, we hypothesized that CD4 T cell recognition J. Lindsay2, T.T. MacDonald1 & A.J. Stagg1 of the M2e epitope conferred a better level of protection than specific 1Centre for Immunology and Infectious Disease, 4 Newark St, London, serum antibody levels alone. Next we analyzed the TCR-repertoire UK, 2Centre for Digestive Diseases, 4 Newark St, London, UK that recognized M2e and investigated the type of T cell response induced subsequent to immunization. The CD4 T cell response was Background: Control of T-cell reactivity with the human intestinal oligoclonal and the recall response to M2e in immunized Balb/C mice mucosa is poorly understood. Type I Interferon (T1IFN) signals via was dominated by IL-17. In addition, vaccinated IL-17 deficient mice JAK/STAT, particularly STAT1, and supports Treg function in showed reduced survival following live challange, indicating involve- mouse models of colitis. T1IFN has been used as a treatment in ment of Th17 M2e-specific CD4 T cells in protection against influ- IBD. We therefore hypothesised that constitutive T1IFN had a regu- enza. The M2e specific T cells were present in low frequencies in latory role in human intestinal T cells. draining lymph nodes, spleen and lung for more than 12 month after Methods: Endoscopic biopsies or resection specimens were frozen i.n. immunizations and upon re-encounter with antigen they showed for immunohistochemistry (IHC) or cultured in the presence of neu- exceptional ability to expand and protect mice from a lethal influenza tralising anti-IFNb or isotope control. Cells were harvested, stimu- dose. Moreover, a significant increase in anti-influenza hemagglutinin lated with anti-CD3/CD28 antibodies and analysed for cytokine (HA) IgG antibody levels was found in serum subsequent to live chal- production by intracellular staining and by multiplex ELISA of cul- lenge of nude mice that have been reconstituted with memory influ- ture supernatants. Phosphorylated STAT1 was measured by flow enza-specific CD4+ T cells, suggesting that those M2e memory T cells cytometry with or without prior T1IFN stimulation. Colonic sections could provide B cell help to unrelated na€ıve B cells. were stained with ant-IFNb and analysed using fluorescent IHC. The CTA1-3M2e-DD fusion protein is, thus, a highly promising + CD3 T-cells were FACS sorted and expression of Interferon Stimu- mucosal vaccine candidate that promotes both antibody and CD4 T lated Genes (ISGs) and SOCS 1 and 3 determined by q-RT-PCR. cell M2e-specific immunity, both contributing to protection against Results: IFNb was detected in the lamina propria of control and live influenza virus infection. Our study highlights the importance of IBD tissue and ISGs (MXA and 250AS) were expressed by intestinal CD4 T cells for protection against influenza infections. T cells. In vitro, IFNb neutralisation reduced the frequency of pSTAT1+ intestinal T cells (n = 6, P = 0.05) and, in healthy controls, decreased the proportion of IL10-producing intestinal T cells (n = 8, P = 0.01). There was a trend for more IFNc-producers and 019 IFNc levels in supernatants were significantly increased (n = 10, Cyclosporine A treatment of T cells induces apoptosis and P = 0.016). In IBD, intestinal T cells were more responsive to IFNb, decreases the production of IL-13 in ulcerative colitis as assessed by ISG induction, (n = 10, P < 0.03) and pSTAT1 was S. Steiner, M. Neurath, R. Atreya & B. Weigmann increased in T-cells isolated from IBD patients (n = 30 IBD, 16 con- Medical Clinic 1, University Clinic Erlangen, Germany trol, P = 0.03). Concordantly, SOCS1 expression was decreased in IBD samples compared to controls (n = 8 IBD, 6 control), Introduction: Inflammatory bowel disease (IBD) is a chronic inflam- P = 0.047). In contrast to controls, neutralisation of IFNb in IBD mation of the digestive tract caused by a dysregulated immune samples led to a generalised increase in cytokine production, with an response. The two major forms of IBD are ulcerative colitis (UC) and increase in T cells producing all cytokines examined (IL-10, IFNc, Crohn´s disease (CD). The immunosuppressive drug cyclosporine A IL-17 and TNFa, n = 10). (CsA) is a potential rescue treatment to avoid colectomy in severe ste- Discussion: T1IFN is present in the human intestinal mucosa and in roid-refractory UC patients, whereas CsA treatment has no beneficial health may have a regulatory role via T cell production of IL-10. effect in CD patients. The molecular mechanism of action of CsA in There is increased responsiveness of the T1IFN pathway in T cells UC is nevertheless incompletely understood. The aim of this study from IBD patients, associated with a generalised suppression of cyto- was to investigate the effect of CsA on a possible modulation of cytokine production. Thus, T1IFN effects are context dependant, which kine production and apoptosis induction by peripheral blood mono- may explain differing clinical effects of therapeutic T1IFN and nuclear cells (PBMCs) of controls and patients with UC or CD. potentially responses to other environmental factors. Methods: Human PBMCs were isolated from whole blood samples and cultured with anti-CD3/CD28 antibodies in the presence or absence of CsA. Supernatants were taken for analysis of cytokine production (TNFa, IFNg, TGFb IL-4, IL-5, IL-10, IL-13, IL-17A,

IL-17F) by ELISA assays. To determine apoptosis induction FACS 022 analysis for AnnexinV staining and fluorescent stainings for CD4 Depletion of regulatory T cells in the APCmin/+ mouse model of and TUNEL were performed. Additionally, expression of caspase-8, colon cancer enhances T cell recruitment and Th1 associated Bcl-xL, Bcl-2 and Bax were analysed by qPCR. responses Results: It was found that levels of IFNc remained unaffected after P. Akeus1, V. Langenes1, A. von Mentzer1, T. Sparwasser2, the addition of CsA. In UC patients, levels of IL-13 were significantly S. Raghavan1 & M. Quiding-Ja¨rbrink1 reduced after CsA treatment in vitro, whereas in PBMCs of controls 1Microbiology and Immunology, Box 435, G€oteborg, Sweden, 2Institute and CD patients IL-13 production was unaffected by CsA treatment. for Infection Biology, TWINCORE, Hannover, Germany Levels of TNFa, IL-10 and IL-17A were significantly reduced after CsA treatment in PBMCs of all patients groups. TGFb and IL-4 pro- Regulatory T cells (Treg) are important to prevent autoimmunity, duction was significantly decreased with addition of CsA in controls, microbial driven inflammation and allergy. In colorectal cancer, but was unaltered in UC and CD patients. Controls showed a signifi- Treg infiltration is associated with a better patient outcome, in cant reduction of IL-5 and IL-17F production after CsA treatment. contrast to most other tumors. We have previously demonstrated Levels of IL-5 were also significantly decreased in UC patients in the that Treg from cancer patients profoundly reduce transendothelial presence of CsA, whereas levels of IL-17A were significantly reduced migration of conventional T cells. To evaluate if Treg also affect in CD patients. Additionally, cultured CD4+ PBMCs of controls and lymphocyte infiltration into intestinal tumors, we have used APC- UC patients underwent apoptosis which was induced independent of min/+ mice that spontaneously develop intestinal tumors due to a the mitochondrial apoptosis pathway, but dependent on activated mutation in the APC tumor suppressor gene. The frequencies of caspase-8 signalling. FoxP3+ putative Treg, CD4+, and CD8+ T cells were determined Conclusion: IBD patients, suffering from Th2 associated UC, can be by flow cytometry and immunoflorescence and these analyses treated successfully with CsA. This study demonstrates for the first revealed an accumulation of Treg and a decrease of conventional time that CsA selectively induces apoptosis in CD4+ blood cells of T cells in the tumors. To evaluate if Treg actively inhibit lympho- UC patients in a mitochondrial independent way associated with a cyte migration into tumors, APCmin/+ mice were crossed with diminished production of the Th2-like cytokine IL-13. DEREG mice, in which Treg can be selectively depleted with diphteria toxin. Short term Treg depletion in tumor-bearing mice resulted in increased frequencies of conventional T cells in the tumors, and an increased frequency of proliferating (Ki67+)T 020 cells, suggesting that the increased T cell frequencies may at least Deletion of IL-4Ra on Macrophages renders mice resistant to partly result from increased local proliferation in the tumors. Treg intestinal inflammation depletion also resulted in a strong up-regulation of mRNA for the Th1 associated chemokines CXCL9 (P < 0.01) and CXCL10 G. Pickert (P < 0.001) specifically in the tumors. In parallel, expression of Institute of Translational Immunology, Oberezahlbacher strasse 63, the corresponding chemokine receptor CXCR3 was increased on Mainz, 55131, Germany conventional T cells migrating into the tumors after Treg deple- Background and Aims: The inflammatory bowel diseases (Crohn’s tion. Depletion of Treg also resulted in increased T cell produc- disease (CD) and ulcerative colitis (UC)) are illness characterized by a tion of IL-17 and IFN-gamma in the tumors. In conclusion, Treg chronic clinical course of relapse and remission associated with self- depletion results in increased accumulation of conventional T destructive inflammation of the gastrointestinal tract. In both UC and cells, especially Th1 associated CXCR3+ T cells, in intestinal CD, leukocyte infiltration into the intestine is fundamental event in tumors and targeting of Treg as an anti-tumor immunotherapy disease development and progression. Th2 cytokines IL-4 and IL-13 may thus improve not only effector functions of activated T cells, are known susceptibility factors for IBD and induce their biological but also their recruitment to tumors. functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ra). We investigated whether activation of interleukin IL-4Ra systemic and on macrophages determines their effector functions and mediates intestinal inflammation in experimental mice model. 023 Methods and Results: We studied the role of IL-4Ra by using the sys- Dissecting the role of intestinal epithelial barrier in the temic IL-4RaKO and macrophage-specific IL-4RaKO (IL-4RaLysM) pathogenesis of ulcerative colitis mice in a mouse model of dextran sodium sulphate induced colitis. I. Dotti1, N. Planell1, R. Mora-Buch1, P. Jung2, E. Batlle2, J. Panes1 Mice were challenged with 2.5% DSS for 14 days to induce colitis. & A. Salas1 a DSS-treated systemic and macrophage specific IL-4R KO mice were 1IDIBAPS, Rossello 149-153, Barcelona, Spain, 2IRB, Baldiri Reixac 10, resistant and showed protection to DSS-induced intestinal inflamma- Barcelona, Spain tion compared to WT mice. IL-4RaKO and macrophage-specific IL- 4RaKO (IL-4RaLysM) mice have no/less inflammation, indicating Ulcerative colitis (UC) is a chronic inflammatory bowel disease no/less weight lost and no epithelial erosions compared to WT mice. (IBD) of unknown aetiology. Alterations in the colonic epithelial Resistance to DSS-induced inflammation in IL-4RaKO/IL-4RaLysM barrier contribute to the onset of UC. However, little data is avail- mice correlated with reduced numbers of inflammatory cells like F4/ able about the primary role of epithelial cell (EC) dysfunction in this 80 and Neutrophils in the colonic mucosa and showing well-struc- disease. Recently, a novel cell-culture system has been established to tured epithelial cell layer without any damage. study gastrointestinal EC physiopathology. Conclusions: IL-4Ra plays an important role in inflammatory bowel Using this approach we aim to investigate whether a primary disease. Deficiency of IL-4Ra systemic and macrophage-specific IL- defect in intestinal EC function is present in UC patients that could 4RaLysM mice reduces intestinal inflammation in a mouse model of drive the development of an inflammatory response. DSS induced colitis. Decreased inflammation is accompanied by lack To this end, we collected biopsies from the sigmoid colons of 6 of proinflammatory immune response. UC patients and 4 non-IBD controls. Isolated crypts were cultured and stem EC were expanded in Matrigel as “organoids”. Total RNA from stem and differentiated organoids was extracted for transcrip- tional analysis.

Our results show that control and UC organoids follow common adulthood. To characterize the adverse effect of early stress on differentiation programs with comparable downregulation of stem immune system development, we analyzed the consequences of and proliferation markers (i.e., Lgr5, Ki67) and upregulation of EC maternal separation (MS) in mice on intestinal barrier functions and differentiation markers (i.e., MUC2, ANPEP, ChrA). However, statis- systemic immune response toward microbiota. tical analysis of microarrays revealed more than 800 genes differen- In 50-days old male mice, MS increased intestinal permeability to tially expressed between control and UC organoids, with cellular FITC-Dextran-4 kDa and visceral sensitivity. MS decreased fecal movement and development, lipid metabolism, and molecule trans- lysozyme activity, IgA concentration and ILC3 population. This port the most significantly altered cellular functions. breach in mucosal barrier function was associated with a systemic In the present study the organoid culture model has been success- IgG response against E. coli. Furthermore, MS increased anti CD3/ fully used to detect intrinsic differences in the epithelium of UC CD28-induced IFNc and IL10 secretion in splenocytes. These results patients. Validation experiments are ongoing to confirm our results are in accordance with an increase of CD4+CD44highCD62Llow in an independent cohort of patients and to dissect the functional active T cells and decreased CD4+CD25+Foxp3+ regulatory T cells. meaning of such alterations. Furthermore, MS decreased CD40 expression in CD11c+ and CD11b+ leading to decrease of IL10 and TGFb secretion without any modification of IFNc on E. coli-stimulated splenocytes suggesting a defect of antigenic presentation. No modification (quantitative 024 nor qualitative) of fecal E. coli population was observed at 50 days Early maternal separation induced commensal e. coli in MS mice. overgrowth triggering visceral sensitivity and specific humoral For the first time, we demonstrated that early stressful events response in adult mice impaired intestinal barrier functions leading to an exacerbated A. Riba, M. Olier, C. Lencina, V. Bacquie, C. Harkat, M. Gillet, humoral response toward microbiota. Inflammatory T cell response C. Salvador-Cartier, M. Baron, C. Sommer, V. Mallet, was increased in MS mice but dampened by an antigenic presenta- V. Theodorou & S. Menard tion defect. Adult mice experiencing early stressful events mimic INRA, ToxAlim, 180 Chemin de Tournefeuille, Toulouse, France common symptoms of gastrointestinal disorders and might by an important factor in those pathologies. Small Intestinal Bacteria Overgrowth (SIBO) in favor of Enterobacte- riaceae is highly associated to Irritable Bowel Syndrome (IBS) a functional gastrointestinal pathology where a specific humoral response toward bacterial antigens has been observed. Symptoms of 026 IBS and SIBO are similar consisting in abdominal pain, bloating and Early steps to explore mucosal immune components in a marine altered stool forms. Further, IBS symptoms can be generated and/or vertebrate exacerbated by early life stressful events. In this study, using the well W. E.-D. Aquino1, A. Olvera-Ramirez2 & K. Acevedo-Whitehouse1 described rodent model of IBS: maternal separation, we developed a 1Unidad de Microbiologıa, FCN, UAQ, Queretaro, Mexico, 2Facultad mouse model of SIBO and studied its consequences on IBS symptom de Ciencias Naturales, UAQ, Mexico and on humoral response toward microbiota. MS increased visceral sensitivity and induced ileal and fecal over- Maturation of the mucosal immune system is influenced by the growth of E. coli in adult female mice. This overgrowth was associ- environment, behavior and feeding, and, in carnivores and primates, ated with a decrease of fecal lysozyme antimicrobial activity and the first 12 months of life are critical for its proper development. increase of anti-E. coli IgG and IgA. In order to decipher whether or Regrettably, despite being the frontline for defense, little is known not those alterations were a consequence of E. coli overgrowth, adult about mucosal immunity of free-living mammals, and even less is mice were force fed daily with 109 commensal E.coli for 15 days. E. known about its ontogeny and ecological constraints. In this context, coli gavage reproduced ileal and fecal overgrowth as well as visceral we have used the California sea lion, a long-lived, sexually-dimor- hypersensitivity and increased anti-E. coli IgG and IgA. However, E. phic, amphibious top predator with well-characterized life stages, in coli gavage didn’t decrease lysozyme antimicrobial activity in feces an attempt to understand the early development of mucosal immu- suggesting that lysozyme defect is a consequence of MS that can par- nity and to investigate the contribution of ontogenic and energetic ticipate to the E. coli overgrowth. traits on expression of mucosal immune components. At Granito This study shows for the first time that (i) maternal separation in Island (Gulf of California), we collected rectal, genital, nasal and oral mice weakened intestinal antimicrobial defense that might result to swabs from 2-, 5- and 12-month old pups. Body condition and vari- intestinal E. coli overgrowth and (ii) intestinal E. coli overgrowth ous health parameters were determined for all. SDS-PAGE gels were was responsible for visceral hypersensitivity and systemic humoral run on extracted proteins to characterize the profile of each mucosa. response toward microbiota. Distinct band patterns were evident between mucosal types and between age classes. Genital mucosa proteins differed between sexes (F = 46.32, gdl= 41, P < 0.001), specially at 5-months of age. For all stages of development, the anal mucosa showed the great diversity of 025 proteins (~100) and widest molecular range. Some bands were only Early maternal separation leads to abnormal immune response found at 2- and 12-month old pups (84 and 26 kDa, respectively). against commensal microbita in adult mice Protein sequencing and immunodetection techniques will now allow A. RIiba, C. Lencina, V. Bacquie, C. Harkat, M. Gillet, us to characterize the proteins, identify those with immune function, C. Cartier-Salvador, M. Baron, C. Sommer, V. Mallet, M. Olier, and explore their ontogenic and energetic constraints. V. Theodorou & S. Menard INRA, ToxAlim, 180 Chemin de Tournefeuille, Toulouse, France Neonatal period is characterized by immature intestinal epithelium and immune system. Occurrence of adverse events during this period induces long lasting alterations of intestinal homeostasis associated with susceptibility to develop gastrointestinal disorders at

027 Enterocytes represent 80% of cells constituting the intestinal epithe- Effect of “acid protect” supplementation on the volatile faecal lium. These cells act like a barrier and contribute to the local metabolome of thoroughbred racehorses immune responses by secreting several soluble factors. Under inflammatory conditions, the number of non-polarized enterocyte stem N. Larkins1, C.J. Proudman2, J. Travers3, C.J. Probert3 & cells increases due to the stimulation of the cell renewal process. K. Koare1 In this context, we proposed to analyse the effects of certain pro- 1Nutritional Laboratories, Monmouth, UK, 2School of Veterinary biotic bacteria using an intestinal epithelial culture model, Caco-2 Medicine, University of Surrey, Gulidford, Surrey, UK, 3Department of cells. These cells were culture for 7 days (non-polarized and undif- Gastroenterology ferentiated cells) and for 21 days (polarized and differentiated cells) The importance of the intestinal microbiome to mammalian health and stimulated with probiotics at different concentrations. We analy- is now widely recognised. Intestinal bacterial communities not only sed the changes on chemokine expression, trans-epithelial electrical contribute to digestion of food material but are important partners resistance (TEER), distribution of zonula occludens (ZO)-1 by in host-microbial co-metabolism. Studies in laboratory animals and immunofluorescence, and the bacterial capacity of adherence. We in humans have demonstrated profound effects of altered gut micro- found that non-polarized Caco-2 cells were more sensitive to bacte- bial communities on many facets of mammalian health including rial stimuli than fully polarized Caco-2 cells in chemokine expres- immunity, behaviour, heart disease and obesity. As a hindgut fer- sion. Moreover, the probiotic with the higher effect on chemokine menter the horse is likely to be even more reliant upon its intestinal expression on non-polarized Caco-2 cells triggered an increase in the microbiome than many other species. Faeces are easily sampled from monolayer’s TEER, in agreement with the fact that a slight increase horses and offer a diagnostic window into the structure and function in pro-inflammatory response could reinforce the epithelial mono- of gut microbial communities. The volatile faecal metabolome, not layer. We cannot confirm a relationship between the distribution of previously investigated in the horse, may offer opportunities for ZO-1 and TEER´s increase, however we observed some membrane monitoring equine intestinal health. changes in bacteria-stimulated Caco-2 cells. At the highest concen- The aim of this study was to identify changes in faecal volatile tration, some probiotics induced damage to the epithelial mono- metabolome in Thoroughbred racehorses after supplementation with layer. Acid Protect In summary, there are probiotics with no effect on the expression The study population of 8 Tb racehorse was selected using the of pro-inflammatory chemokines and these could be useful in the following criteria: maintenance of intestinal homeostasis, whereas others may have a * Male and castrated role to prevent damage to the epithelial monolayer. * Over 2 years of age * In race training * Healthy at the beginning of sampling * No medication administered in previous 3 months 029 Fresh faecal samples were collected prior to exercise and frozen at Effects of selective PI3Kd inhibitors on activation and -80C prior to analysis. After the first two faecal samples were col- downstream signalling in T cells lected horses received dietary supplementation with 75 g (tid) for K. Bjorhall,€ S.R. Blomqvist, L. Yrlid, M. Gustavsson, M. Uddin, 14 days. N. Holden & K. Karabelas Samples were thawed, warmed in a water bath to 38 C for AstraZeneca RIA iMED, Pepparedsleden 1, M€olndal, Sweden 20 minutes, and volatile organic compounds (VOCs) adsorbed onto a solid phase adsorption fibre for 30 minutes. VOC chromatograms The Class I phoshoinositide-3-kinase delta (PI3Kd) isoform regulates for each faecal sample were obtained by thermal desorption from the essential immune functions in a wide variety of cells, including leu- fibre followed by separation by gas chromatography and identifica- kocytes. Its importance in inflammation signalling, particularly in B tion by mass spectrometry (GC-MS). Chromatograms were inter- and T cells, has stimulated the clinical development of selective preted by both manual and automated readings to identify and PI3Kd inhibitors. As Th2-driven allergic airway inflammation plays quantify VOCs present. an important role in the pathogenesis of asthma, our aims was to Conclusions: evaluate the effects of a selective PI3Kd inhibitor on T cell activation * We have demonstrated the potential utility of faecal VOC analy- in an ovalbumin (OVA)/ anti-CD3-induced airway inflammation sis by GC-MS for characterisation of the intestinal metabolic envi- model in transgenic OT.2 mice in vivo. ronment. We have developed highly selective PI3Kd inhibitors that block * We demonstrate repeatable VOC profiles from similarly man- the release of IL-5 in human PBMCs and IL-17 by human Th17 aged horses. cells, in a concentration-dependent manner in vitro. To elucidate the * Our analysis can detect small shifts in VOC profile possibly pharmacodynamic effects of our PI3Kd inhibitors on effector/mem- associated with dietary supplementation. ory T helper cells in the lung, we used OVA specific, MHC class II * Increase in butanoic acid in post-supplementation samples is of restricted ab T-cell receptor (TCR) OT.2 transgenic mice. Upon particular interest as this compound is associated with enterocyte local OVA provocation challenge, T-cells were activated and health. recruited into the lungs. Five days later, the OT.2 mice were given an intranasal (i.n.) anti-CD3 challenge and the downstream effects on PI3K signalling were measured through phosphorylation of S6 ribosomal protein (pS6RPSer235/236), cytokine release and T cell 028 activation markers (CD62L, CD69 and CD25) in the absence and Effect of six strains of probiotics on the integrity of a model of presence of a selective PI3Kd inhibitor. intestinal epithelial monolayer We found that pS6RPSer235/236 were significantly downregulated in CD4+T cells by our PI3Kd inhibitors 6 h after anti-CD3 challenge B. Martınez-Abad1, E. Montalvillo1, C. Escudero-Hernandez1, (P < 0.002). However, no significant effects on activation markers J.A. Garrote2 & E. Arranz1 (CD62L, CD69 and CD25) on CD4+ T cells or on cytokine levels in 1Pediatris & Immunology, C/Ramon y Cajal 7, Valladolid, Spain, bronchoalveolar lavage fluid were observed at the same time- 2Genetics Lab. Hospital U. Rio-Hortega, Dulzaina 2, Valladolid, Spain point.The mechanism underlaying this dissociation in unclear.

Our results show that S6RP is phosphorylated at Ser235/236 in of SIgA uptake by M cells are now well known and described, the CD4+ T cells after anti-CD3 challenge and that this phosphorylation mechanisms whereby SIgA is selectively bound and taken up remain is inhibited by selective PI3Kd inhibitors. We therefore conclude that poorly understood. Here we first demonstrate that both the Ca1 pS6RPSer235/236 can be used as a target engagement marker reflect- region and glycosylation, more particularly sialic acid residues, are ing PI3Kd activity in this in vivo model. involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1+ 030 dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that Epithelial IL-1R2 acts as a homeostatic regulator of mucosal and systemic antibody responses against the HIV p24-SIgA inflammatory signals during remission of ulcerative colitis complexes administered orally is strictly dependent on the expression R. Mora-Buch1, I. Dotti1, N. Planell1, E. Calderon-G omez 1, of Dectin-1. Having deciphered the mechanisms leading to specific P. Jung2, M.C. Masamunt1, E. Batlle2, J. Panes3 & A. Salas1 targeting of SIgA-based Ag complexes paves the way to the use of such 1IDIBAPS, Rossello 153, Barcelona, Spain, 2IRB, Baldiri Reixac 10, a vehicle for mucosal vaccination against various infectious diseases. Barcelona, Spain, 3Hospital Clınic de Barcelona, Villarroel 170, Barcelona, Spain Ulcerative colitis (UC) is a chronic colonic disease that presents peri- 032 ods of active inflammation followed by periods of remission. We Evaluation of immunogenicity and protective efficacy of Type III focused on indentifying regulatory mechanisms that favor intestinal Secretion System proteins of enteropathogenic bacteria homeostasis during disease remission. A total of 180 UC patients B. Jneid, M. Leonetti, P. Lamourette, K. Moreau, (UC active or remission) and 55 healthy non-IBD controls partici- M. Plaisance, A. Savatier, C. Creminon & S. Simon pated in the study. Using real-time PCR and ELISA of culture super- CEA Saclay, IBITEC-S, SPI, LERI, Gif-sur-Yvette, France natants from colon biopsies, we found that the interleukin-1 (IL-1) decoy receptor gene (IL1R2) and secreted protein (IL-1sR2) were Diarrheal diseases caused by bacterial pathogens e.g. Escherichia coli, significantly (P < 0.05) up-regulated during UC remission compared Salmonella enterica, Shigella flexneri, etc, remain a major public to UC active and control mucosa. In contrast, the IL-1 receptor health problem in developing countries, that contribute to the high antagonist (IL-1Ra), together with IL-1b, IL-1 receptor type 1 (IL- mortality rate, especially for children younger than 5 years [1]. These 1R1) and IL-1 receptor accessory protein (IL-1RAcP), were overex- bacteria have evolved an of molecules that are mostly secreted by pressed in UC active mucosa. Immunostaining identified both lam- dedicated Type III Secretion System (T3SS). Part of the T3SS forms ina propria IgA+ cells and adjacent mucosal epithelium as IL-1R2+. an extracellular needle and syringe necessary to inject effector pro- Nevertheless, we found by intracellular flow cytometry staining teins in the host cell, subverting normal cellular functions and caus- of digested biopsies that epithelial cells were the cellular source ing enteric infections [2, 3]. In contrast to the large diversity responsible for the upregulation of IL-1R2 during UC remission. observed among the effectors proteins [4], the structural proteins Culturing whole colonic crypts or epithelial stem cells, we demon- that compose the injectisome (OM ring, needle and needle tip) are strated that IL-1R2 is negatively regulated by wnt/beta-catenin sig- relatively conserved among the various pathogenic bacteria. Because naling, and is therefore up-regulated during epithelial cell these proteins are required for pathogenesis and share similarities in differentiation. Importantly, IL-1R2 produced by isolated colonic all virulent enteropathogenic bacteria, they are ideal candidate anti- crypts of UC patients in remission, but not controls, reduced IL-8 gens for a subunit-based, broad-spectrum vaccine. We will examine induced by exogenous IL-1b. Furthermore, expression of the IL1R2 the immunogenicity and protective efficacy of the different structural in the remitting mucosa could be a predictive molecule of UC proteins: needle proteins PrgI/MxiH/EscF, needle tip proteins SipD/ relapse during a 1-year follow-up. In summary, we hypothesize that IpaD/EspA and OM ring proteins InvG/MxiD/EscC of S. enterica, S. IL-1R2 represents a homeostatic molecule, whose increased expres- flexneri and E. coli respectively, alone or combined, in a mouse sion during remission may represent an endogenous mechanism that model of infection. Keywords: Enteropathogenic bacteria, Type III could dampen local inflammation and prevent disease relapse. Secretion System, immunogenicity, vaccine.

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Essential role of Dectin-1 in intestinal M cell-mediated reverse Follicle homing antigen presenting cells modulate TH2 bias transcytosis of SIgA-antigen complexes B. Barry, D.S. Donaldson & N.A. Mabbott N. Rochereau1, D. Drocourt2, E. Perouzel2, V. Pavot3, The Roslin Institute, The Roslin Institute Building, Edinburgh, UK P. Redelinghuys4, G.D. Brown4, X. Roblin1, B. Verrier5, The expression of the chemokine receptor CXCR5 by dendritic cells C. Genin1, B. Corthesy6 & S. Paul1 and their homing to B-cell follicles are suggested requirements for the 1Jean Monnet University, 15 rue Ambroise Pare, St-etienne, France, generation of T-helper type 2 (T 2) cells in response to infection. Pre- 2Cayla-InvivoGen, Toulouse, France, 3Institut de Biologie et Chimie des H vious studies revealed that bone marrow chimeric mice deficient in Proteines, Lyon, France, 4University of Aberdeen, Aberdeen, UK, CXCR5 in dendritic cells or CD4+ T-cells impaired the development of 5Institut de Biologie et Chimie des Proteines, Lyon, France, 6R&D both T-follicular helper (T )orT 2 cells after infection with Heligmo- Laboratory of the Division of Immunology, Lausanne, Switzerland FH H somoides polygyrus (Leon, Ballesteros-Tato et al. 2012). Using a refined Intestinal microfold (M) cells possess a high transcytosis capacity and Cre/LoxP conditional gene expression model we have generated a spe- are able to transport a broad range of materials including particulate cific CD11c-mediated CXCR5 knockout transgenic mouse on a C57Bl/ antigens, soluble macromolecules and pathogens from the intestinal 6 genetic background. Characterisation of this model has revealed that lumen to inductive sites of the mucosal immune system. M cells are CD11c+ cells are capable of trafficking to and are restricted within T- also the primary pathway for delivery of secretory IgA (SIgA) to the cell regions of lymph nodes & spleen, and are unable to traffic into the gut-associated lymphoid tissue. However, although the consequences B-cell follicle. Infection with the gastrointestinal nematode Trichuris

muris stimulates a TH2 dominated response in resistant mouse strains 035 such as C57Bl/6 with worm clearance occuring within 21 days. Mouse Gut microbial antigen speciﬁc CD4+T cells from Crohn’s Disease

strains susceptible to T. muris infection display a TH1 dominated patients exhibit a pro-inflammatory Th17 phenotype response and remain persistently infected. We investigated the ability E. Calderoń-Go´mez1, H. Bassolas-Molina1, R. Mora-Buch1, of CD11c-CXCR5-/- mice to mount an appropriate T 2 response to H 1 2 3 4 T. muris infection to facilitate clearance. Unlike CXCR5 fl control I. Dotti , R. Cabezon*, S. Singh , J. Panes , D. Benıtez-Ribas & 1 mice, CD11c-CXCR5-/- mice were unable to clear T. murisinfection A. Salas 1 2 after 30 days. Gene expression analysis of cytokine responses in the IDIBAPS, Rossello 149-153, Barcelona, Spain, Prometheus 3 mesenteric lymph nodes of T. muris-infected mice revealed increased Therapeutics and Diagnostics, San Diego, USA, Hospital Clınic 4 IFNG, IL1B, IL2, IL6, IL10 and reduced IL4, IL9 and IL25 mRNA CIBERehd, Villarroel 170, Barcelona, Spain, CIBERehd, Rossello expression in CD11c-CXCR5-/- mice compared to CXCR5 fl control 149-153, Barcelona, Spain mice. These alterations in cytokine expression were associated with Experimental models have led to the theory that chronic inflamma- increased expression of both IL-12 receptor beta subunits, IL12RB1 tion, as seen in Crohn’s disease (CD), results from a loss of tolerance and IL12RB2, and the co-stimulatory molecules CD80 and CD86. We towards commensal microbiota. In fact, antibodies specific for + have demonstrated that CXCR5 deficiency in CD11c cells alters the microbial components are found in 50% of CD patients, indicating ability to form a coherent TH2 type response to T. murisinfection, pre- that a memory T-cell response might be generated as well. There is venting worm clearance. These data confirm that for the efficient for- yet little evidence, however, in human disease proving the later. In mation of a TH2 response to infection with intestinal nematodes, testing reactivity to several gut microbial antigens in peripheral + CD11c cells are required to localise to the B-cell follicle via expression blood, we have detected T-cell responses towards antigens including of the chemokine receptor CXCR5. Leon, B., A. Ballesteros-Tato, et al. FlaX, Fla2 and YidX in both healthy individuals and CD patients. + (2012). “Regulation of TH2 development by CXCR5 dendritic cells Interestingly, the proliferative response was significantly higher and lymphotoxin-expressing B cells.” Nat Immunol 13(7): 681-690. among CD patients compared to controls, which also correlated with an increased production of IFN-gamma and IL-17 as assessed by ELISA. Intracellular cytokine staining showed the presence of Th17, 034 Th1 and Th17/Th1 cells among the FlaX, Fla2 and YidX-specific T- cell populations, whose frequency was higher in CD. Furthermore, Fossil fuel derived ambient particulate matter (pm10) induces real time-PCR analysis of RORc, IL-17A and IL-17F expression on multiple pathways of activation in human dendritic cells (dc) sorted FlaX, Fla2 and YidX-specific CD4+T cells revealed a clear bias W. Martha, N. Mushtaq, M.N. E, W. Abigail, B. Rossa, towards a pathogenic Th17 phenotype only in CD patients’ T J. Sanders Theodore, M. Grigg Jonathan & A.J. Stagg cells, but not in T cells from healthy controls. Thus, our data indi- The Blizard Institute, Queen Mary, University of London, London, UK cate that T cells that react to the same gut microbial antigen have been differently imprinted with a pro-inflammatory phenotype dur- Fossil fuel derived particulate matter (PM) is present within airway ing CD. We hypothesize that these circulating memory T cells may myeloid cells of individuals exposed to pollution and is linked with contribute to sustain gut inflammation in CD upon antigen re- respiratory and systemic inflammatory disease. We hypothesize that encounter; their identification opens new avenues for antigen-direc- exposure of airway DC to fossil fuel PM enhances their ability to ted therapies in CD. induce inflammatory responses. Therefore, we examined the effects of urban ambient PM of <10 µm (PM10) on human monocyte derived DC (MoDC) and on ex vivo respiratory tract DC (RT-DC) FACS sorted from induced sputum. DC phenotype was assessed by flow cytometry and qRT-PCR; stimulatory capacity determined in a mixed leukocyte reaction. 036 Like the TLR4 agonist LPS, PM10 induced ‘classical’ DC activa- High fat feeding alters gut microbiota and protects mice from tion by dose-dependent up-regulation of MHC class II, CD40, CD86 colitis and colitis-associated colorectal cancer and CCR7 as well as the production of IL-6 and IL-12p70. In accor- S. Melgar1, C. O’Mahony1, S. Clarke1, G. Hurley1, A. Gavin1, dance with their mature phenotype, PM10 treated DC were more 1 1 2 2 1 + A. Quinlan , A. Houston , A. Mahmoud , M. Bennett , P. Cotter stimulatory for naive CD4 T cells. In contrast to LPS, PM10 also & F. Shanahan1 induced the release of cytokines associated with inflammasome acti- 1Alimentary Pharmabiotic Centre, Cork, Ireland, 2Cork University b vation (IL-1 and IL-18) and IL-23. PM10 stimulation, but not LPS, Hospital, Cork, Ireland additionally induced aryl hydrocarbon receptor (AhR) signalling in MoDC as indicated by AhR-dependent induction of the target gene Inflammatory Bowel Disease (IBD) appears to be triggered by envi- CYP1A1. PM10 also induced CYP1A1 in ex vivo RT-DC. Carbon ronmental factors (e.g. diet, microbiota composition) leading to dys- black particles, representing the particulate core of PM, did not acti- regulated immune responses in genetically susceptible individuals. vate DC indicating a critical role for other components. To date, studies exploring the cross-interaction between diet, Thus, fossil fuel derived PM10 induce a complex programme of immune responses and microbiota in the pathology of IBD are lim- DC activation that includes classical maturation, inflammasome- ited. A recent study reported that a diet based on milk derived fat dependent cytokines and AhR signalling. How these pathways inter- led to the expansion of a specific pathobiont resulting in worsened act to influence DC function and immune regulation in the human colitis in IL-10-/- mice. In this study, we examined the temporal airway is under investigation. relationship between diet, the gut microbiota and immune responses in experimental models of Colitis and Colitis-associated Cancer (CAC). Mice were fed lard-based high fat diet (HFD-45%Kcal) or low fat diet (LFD-10%Kcal), followed by AOM injection and 3x DSS-cycles [(1.5%DSS-5 days & water-14 days), CAC-model] or 3xDSS-cycles (Colitis-model). HFD-feeding protected mice from developing colitis and CAC as demonstrated by reduced tumour incidence and numbers, improved colon length, body weight, and

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 23 histological scores and reduced plasma and colonic/tumor inflamma- colitis. Our aim was to investigate the role of the mucus barrier in tory mediators. Mice with Colitis and CAC fed with HFD presented small intestine by comparing age-dependent biological responses in an improved microbial diversity, associated with a reduction in Pro- mice with deficient Muc2 production. teobacteria and an increase in Firmicutes and Verrucomicrobia com- Materials and Methods: Muc2+/+, Muc2+/- and Muc2-/- mice were pared to their LFD-fed counterparts. Collectively and contrary to the reared under SPF conditions and sacrificed at 2, 4, and 8 weeks of previous report using a milk fat based diet, we show that lard-based age. Total RNA from ileum and colon was purified, and analysed for HFD alters the gut microbial populations protecting against colitis full genome transcriptome analysis. Microbiota composition of faecal and reducing the risk for CAC. The data highlights a far more com- samples was determined using a mouse intestinal chip (MITChip). plex regulation of lard-based HFD on microbiota composition and Morphological and immunohistological studies were performed on host immune responses warranting further investigations on lard- segments of ileum and proximal colon. components and other diets as triggers IBD and CAC pathology. Results and Discussion: Muc2-/- mice develop colitis in proximal Coliti-associated cancer; high fat diet; microbiota; colitis colon after 4 weeks, which worsens by week 8, as evidenced by progressive mucosal thickening, epithelial morphological changes and bacterial invasion. No overt morphological changes were observed in ileum, apart from longer villi due to hyperproliferation in the 037 Muc2-/- mice at weeks 4 and 8. Muc2 expression increased with age Histone acetylase inhibitors induce caspase-1 independent IL-1b in mice+/+; in Muc2-/+ mice expression was half that of Muc2 + /+ secretion mice. Innate immune responses were elevated in ileum in Muc2-/- + D. Stammler1, E. Tatjana1, M. Sarah2, J.-S. Frick2, A. Dalpke1, and Muc2-/ at weeks 4 and 8. By week 4 and 8, adaptive immune ^ K. Heeg1 & K.A. Bode1 responses and inflammatory signalling pathways, including NF-eB + 1Department of Infectious Diseases -Med. Microbiol, Im Neuenheimer activation, were down-regulated in Muc2-/- and Muc2 /-. Lipid Feld 324, Heidelberg, Germany, 2Institute of Medical Microbiology Uni. metabolism pathways were down-regulated in ileum at weeks 4 and Tubingen,€ Elfriede-Aulhorn-Str. 6, Tubingen,€ Germany 8 suggesting altered metabolic functions. Colonic microbial composition is different in Muc2-/- compared to Muc2 + /+, whereas the In the course of the last years evidence has accumulated showing that differences are less obvious in ileum. In colon, Muc2 deficiency leads histone deacetylase inhibitors (HDACi) have important immune mod- to inflammation and tissue damage. In contrast, there are less histo- ulatory activity. In the present work it is shown that in human and logical changes in ileum despite increased cytokine and chemokine murine dendritic cells and murine macrophages HDACi are strong expression, and decreased expression of inflammatory pathway genes. b activators of LPS induced IL-1 processing and secretion. Strikingly, In ileum, microbiota composition, lower total numbers of bacteria, b this IL-1 secretion was independent of the inflammasome compo- and/or intrinsic homeostatic mechanisms appear to prevent tissue nents NLRP3, ASC and even caspase-1 and activation kinetics differed damage in Muc2-/-. Muc2+/- mice are a good model to study the completely from that observed after inflammasome activation. Inhibi- role of mucus in intestinal health and have implications for IBD tion studies showed that the histone deacetylase HDAC10 is responsi- where mucus levels are reduced during active disease and remission. ble for this HDACi/LPS induced IL-1b secretion. Mechanistically, HDACi/LPS induced IL-1b secretion was strictly dependent on Trif and was associated with a functional impairment of autophagic processes. Importantly, these data demonstrate that besides the conven- 039 tional inflammasome dependent IL-1b cleavage, dendritic cells and How can differentially acting ADP-ribosylating toxins effectively macrophages are capable of activating IL-1b by a novel, alternative prime CD4 T cells and affect germinal center size and function? mechanism. Treatment of mice with HDACi during the induction of a R.K. Chandode, L.F. Yrlid & N.Y. Lycke dextran sulfate sodium-induced colitis resulted in a strong increase of Department of Immunology and Microbiology, Medicinaregatan 7 a, intestinal IL-1b. As naturally occurring HDACi like butyric acid are Gothenburg, Sweden physiologic components of the intestinal milieu, HDACi induced IL- 1b may have a physiological function in intestinal homeostasis. The ultimate aim of vaccination is to induce long term protective Altogether the data demonstrate that in addition to the conven- immunity. Accumulating evidence support a critical role of the choice tional inflammasome dependent IL-1b activation dendritic cells and of adjuvant for an effective stimulation of immunological memory. macrophages are capable to activate IL-1b by a novel until now While cholera toxin (CT) is a potent adjuvant also pertussis toxin (PT) unknown additional mechanism. host adjuvant functions, albeit it is better known to inhibit leukocyte migration. Both toxins are ADP-ribosylating enzymes, but act on two different G-proteins, Gsa and Gi, in targeted dendritic cells (DC). While CT acts on Gsa and stimulates adenylate cyclase, leading to 038 increased intracellular cAMP-levels, PT also promotes intracellular Homeostatic mechanisms prevent ileitis in mice with deficient cAMP-levels, but only indirectly. We have undertaken extensive studies MUC2 production of the adjuvant effect of CTA1-DD, a gene fusion protein that exploits B. Sovran1, L. M.P. Loonen1,P.Lu2, E. Kranenbarg3, C. Belzer4, the enzymatic activity of CT, combined with a DC-targeting D-dimer M. Boekschoten5, P. van Baarlen1, M. Kleerebezem1, P. de Vos6, from Staphylococcus aureus protein A. Unexpectedly, targeted DC J.M. Wells1, I. Renes7 & J. Dekker1 failed to show increased cAMP--levels, while the adjuvant effect clearly 1Host Microbe Interactomics, Wageningen, Netherlands, 2Amsterdam required Gsa in DC. However, the corresponding S1-DD fusion pro- Medical Center, Amsterdam, Netherlands, 3Host Microbe Inetractomics, tein from PT was found to host potent adjuvant effects and similar to Wageningen, Netherlands, 4Microbiology, Wageningen, Netherlands, CTA1-DD intranasal immunizations stimulated strong specific muco- 5Division of Human Nutrition, Wageningen, Netherlands, 6Department sal IgA and systemic IgG responses. Both CTA1-DD and S1-DD of Pathology & Medical Biology, Groningen, Netherlands, 7Department induced enhanced germinal center (GC) reactions and primed CD4 T c of Pedriatric, Rotterdam, Netherlands cells to produce stronger IL-10, IL-17 and INF- levels. Here we have asked to what extent cAMP is involved and which signaling pathways Introduction and Objectives: Muc2 is the major component of the mediate the adjuvant functions of CTA1-DD and S1-DD by exploring intestinal mucus layer, and Muc2-/- mice spontaneously develop a mouse model with a distinct defect of Gsa in the DC cell subset.

040 CD45+CD3+CD4-Valpha7.2+CD161high MAIT cells showed some- Human buccal epithelium acquires microbial what higher frequencies in tumor than unaffected colon tissues hyporesponsiveness at birth, a role for secretory leukocyte (0.72 Æ 0.17%, versus 0.43 Æ .11% of CD45+ leukocytes, P < 0.05), protease inhibitor but there was no correlation between MAIT cell frequencies and tumor stage. The large majority of colonic MAIT cells are recently activated C.L. Menckeberg1, J. Hol1, Y. Simons-Oosterhuis1, memory/effector cells based on their expression of CD69 and CD45RO. H.C. Raatgeep1, L.F. de Ruiter1, D.J. Lindenbergh-Kortleve1, The majority of MAIT cells in unaffected colon tissues (77 Æ 15%, A.M. Korteland-van Male1, S. El Aidy2, P. P.E. van Lierop1, meanÆSEM) produced IFN-gamma, while only 3 Æ 2% produced IL- M. Kleerebezem2, M. Groeneweg3, G. Kraal4, 17. Colonic MAIT cells also produced TNFalpha (29 Æ 9%) and IL-2 B.E. Elink-Schuurman5, J.C. de Jongste5, E. E.S. Nieuwenhuis1 & (27 Æ 7%), as well as Granzyme B (GrB; 49 Æ 6%). In the tumors, sig- J.N. Samsom1 nificantly lower frequencies (P < 0.01) of IFN-gamma-producing 1Laboratory of Pediatrics, Erasmus MC, Rotterdam, Netherlands, MAIT cells were seen (49 Æ 9%), while there were no significant differ- 2Laboratory of Microbiology, Wageningen University, Wageningen, ences in the frequencies of IL-17-, TNFalpha-, IL-2-, and GrB-produc- Netherlands, 3Department of Pediatrics, Maasstad Hospital, Rotterdam, ing cells. In conclusion, we show that MAIT cells are able to infiltrate Netherlands, 4Dept. Molecular Cell Biology and Immunology, VUMC, colon tumors but that their ability to produce IFN-gamma is substan- Amsterdam, Netherlands, 5Department of Pulmonary, Sophia tially reduced. We suggest that MAIT cells have the capacity to promote Children’s Hospital, Rotterdam, Netherlands the local immune response to tumors, but that factors in the tumor Repetitive interaction with microbial stimuli renders epithelial cells microenvironment act to reduce MAIT cell IFN-gamma production. (EC) hyporesponsive to microbial stimulation. Previously, we have reported that buccal EC from a subset of pediatric Crohn’s disease patients are not hyporesponsive and spontaneously released chemokines. We now aimed to identify kinetics and mechanisms of acqui- 042 sition of hyporesponsiveness to microbial stimulation using primary Identification of signaling pathways involved in Rankl-Induced human buccal epithelium. Buccal EC collected directly after birth M cell differentiation using 3-D enteroid cultures and in later stages of life were investigated. Chemokine release and M.B. Wood, D. Rios & I.R. Williams regulatory signaling pathways were studied using primary buccal EC Department of Pathology, 615 Michael St., Whitehead Bldg., Atlanta, and the buccal EC line TR146. Findings were extended to the intesti- GA, USA nal mucosa using murine model systems. Directly after birth primary human buccal EC spontaneously produced the chemokine CXCL-8 Microfold (M) cells are specialized antigen-sampling epithelial cells and were responsive to microbial stimuli. Within the first weeks of restricted to the Peyer’s patches and isolated lymphoid follicles of life these EC attained hyporesponsiveness, associated with inactiva- the small intestine. M cells take up particulate antigens from the tion of the NF-KB pathway and upregulation of the novel NF-KB intestinal lumen and deliver these antigens to the lymphoid follicles inhibitor SLPI but no other known NF-KB inhibitors. SLPI protein beneath the epithelium where adaptive immune responses are initi- was abundant in the cytoplasm and the nucleus of hyporesponsive ated. Previous studies identified RANKL-RANK signaling and the Ets buccal EC. Knock-down of SLPI in TR146-buccal EC induced loss of transcription factor Spi-B as essential mediators for M cell differenti- hyporesponsiveness with increased NF-KB activation and subsequent ation. In the past, it has been challenging to study M cell differentia- chemokine release. This regulatory mechanism extended to the intes- tion in vivo due to their low numbers. We have established RANKL- tine, as colonization of germfree mice elicited SLPI expression in supplemented 3-D enteroid cultures in Matrigel as an in vitro model small intestine and colon. Moreover, SLPI deficient mice had system for studying the process of M cell differentiation. Primary en- increased chemokine expression in small intestinal and colonic EC. teroid cultures derived from wild type mice were stimulated with We identify SLPI as a new player in acquisition of microbial hyp- RANKL at the time of the first subculture. Two days of RANKL oresponsiveness by buccal- and intestinal- epithelium in the first treatment is sufficient to strongly induce multiple M cell-associated weeks after microbial colonization. genes including Anxa5, Spib and Gp2 compared to unstimulated control cultures. These genes are not induced in enteroids derived from conditional knockout mice lacking RANK on intestinal epithelial cells. In other RANKL-responsive cells, RANKL can activate mul- 041 tiple signaling pathways including NF-jB, MAP kinases, PI-3 kinase Human mucosa-associated invariant T (MAIT) cells are present and Src kinase. We used the enteroid model system to test whether in colon adenocarcinomas, but secrete reduced amounts of specific RANKL-activated signaling pathways play a critical role in IFN-gamma M cell differentiation. Inhibitors of MAP kinases, PI-3 kinase and Src kinase did not interfere with RANKL-induced M cell differentia- P. Sundstro¨m1, M. Sundquist1, J. Wong2 & M. Quiding-Ja€rbrink1 tion in C57BL/6 enteroid cultures. No induction of M cell-associated 1Microbiology and Immunology, Sahlgrenska Academy, University of genes was observed in enteroids from aly/aly mice with a nonfunc- Gothenburg, G€oteborg, Sweden, 2Microbiology, National University of tional mutant allele of NF-jB-inducing kinase (NIK). These results Singapore, Singapore pinpoint the noncanonical NF-jB signaling pathway that requires Mucosa-associated invariant T (MAIT) cells are innate-like T cells with NIK as a key proximal event downstream of RANK engagement for a conserved T cell receptor alpha-chain which recognize bacterial intestinal epithelial stem cells to commit to the M cell lineage. metabolites of B vitamins presented on the invariant MR-1 molecule. They are present in intestinal tissues and liver, and they are capable of rapid production of IFN-gamma and IL-17 in response to bacterial insult. In colon cancer, IL-17-driven intestinal inflammation promotes tumor progression, while IFN-gamma-production is associated with improved patient prognosis. However, virtually nothing is known about MAIT cells in human tumors. Here we determined the presence and functional capacity of human colonic MAIT cells, both in colon tumors and tumor-free tissue. Flow cytometric analyses of

043 Immunization via conjunctiva is a new approach to vaccine deliv- IL-17 is required for vaccine induced secretory IgA transport and ery against ocular surface infections. Our results revealed that PmpC protection from Oral Cholera Toxin Challenge has a potential activate type 1 T-cellular immune response, which holds essential for resolving chlamydial infection to higher extent C. McEntee, C. Davitt, A. Abautret-Daly, R.M. McLoughlin & when applied via conjunctiva vs. subcutaneous immunization. E.C. Lavelle School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Dublin 2, Ireland A role for interleukin (IL)-17 in the regulation of polymeric immu- 045 noglobulin receptor (pIgR) expression at mucosal surfaces has Immunogenic effects of a new alpha-gliadin peptide on a recently been described (Jaffar et al, Cao et al). In this study we dendritic cell culture model investigated whether a deficiency in the IL-17 pathway impacted on E. Montalvillo1, C. Escudero-Hernandez1, J.A. Garrote2 & oral vaccine induced IgA responses and susceptibility to oral cholera E. Arranz1 toxin challenge (OCTC). In our experience, three rounds of vacci- 1Pediatrics & Immunology, C/Ramon y Cajal 7, Valladolid, Spain, nation with CT and CTB can induce sufficiently high CTB-specific 2Genetics Lab. Hospital U. Rio-Hortega, C/Dulzaina 2, Valladolid, IgA titres to confer protection against OCTC. Thus, wild-type Spain (WT) C57BL/6 mice and IL-17R-/- mice were vaccinated three times, two weeks apart with CT + CTB and challenged with CT We have previously described a new gliadin peptide released by the two weeks later. Vaccinated WT mice were fully protected from action of gliadin-degrading proteases from the duodenum of celiac CT-induced fluid accumulation following challenge whereas IL- disease (CD) patients. The ability of this peptide to develop a humoral 17R-/- mice exhibited substantial fluid accumulation in the caecum, immune response in vivo in CD patients was confirmed by the detec- indicative of CT enterotoxicity. This susceptibility to OCTC corre- tion of IgA anti-deamidated 8-mer (d8mer) peptide in plasma samples lated with lower levels of faecal CTB-specific IgA in IL-17R-/- mice from these patients. Our aim here was to study the immunogenic when compared to WTs. However, this was not due to a general effects of this peptide in an in vitro model of dendritic cells. Periph- defect in specific IgA production as CTB-specific antibodies in eral blood-derived dendritic cells were obtained from CD patients intestinal tissue were comparable between WT and IL-17R-/- mice. (both active CD, and on GFD) and healthy volunteers. Cell culture for Furthermore, intestinal exposure to CT triggered a very rapid trans- 24 hours was stimulated with 100 lg/ml of each peptide (native 8mer port of IgA into the lumen which was dependent on IL-17, IL-1 and d8mer, d33mer, 19mer, and control peptide). Dendritic cell mat- and the NLRP3 inflammasome. These findings suggest a key role uration and activation markers were analyzed, as well as the ability of for the inflammasome-IL-1-IL-17 axis in CT-induced IgA secretion these cells to induce T cell proliferation in an autologous co-culture. and protective immunity and that IL-17 may be a valuable biomar- Dendritic cells from CD patients stimulated with d8mer show an ker of oral vaccine efficacy. increased expression of CD80 (aCD, P < 0.001, GFD, P < 0.01) and CD86 (aCD, P < 0.01, DSG, P < 0.05); whereas 33mer increased CD83 (aCD, P < 0.001) and CD86 (aCD, P < 0.01, GFD, P < 0.05) expression, compared to the corresponding basal culture. Dendritic 044 cells stimulated with both d8mer and d33mer induced proliferation Immunization via conjunctiva and CALT as a route for chlamydia of IFNc-producing T cells in samples from all CD patients. Both, vaccine delivery: Immune responses to PmpC, an outer 19mer and native 8mer have no effects. membrane protein of C. trachomatis In summary, as with 33mer peptide, the d8mer peptide is involved in the stimulation of dendritic cells and proliferation of IF- A. Inic-Kanada1, S. Belij1, M. Stojanovic2, E. Marinkovic2, Ngamma-producing T cells, probably independent of whether anti- 2 1 1 1 € 1 I. Stojicevic , J. Montanaro , E. Stein , N. Bintner , N. Schurer , gen presentation is mediated HLA-DQ2 + or DQ2 dendritic cells. A. Ladurner1 & T. Barisani-Asenbauer1 1LBCE OCUVAC, Medical University of Vienna, Vienna, Austria, 2Institute of Virology, Vaccines and Sera - TORLAK, Belgrade, Serbia 046 Trachoma is the world’s leading cause of preventable infectious ocular disease. Although antibiotics are effective in treating active cases of the Immunological differences between left and right-sided colonic illness, they do not prevent re-infection, which occurs with high fre- tumours of patients with colorectal cancer quency in susceptible populations. Nevertheless the immunological H.O. Al-Hassi1, G.H. Lee1, G. Malietzis1, D. Bernardo1, mechanisms responsible for protective immunity versus immunopa- E.R. Mann2, D. Reddi1, N.R. English1, R. Man3, R. K.S. Phillips4 & thology are still not well understood, although it is widely accepted that S.K. Clark4 T cell driven IFNc Th17 responses are critical for clearing the infection. 1Immunology, Antigen Presentation Research Group, Watford Road, C57BL/6 mouse strain was immunized via the conjunctiva and London, UK, 2Department of Medicine, Johns Hopkins University, subcutaneously with polymorphic membrane protein C (PmpC), an School of Medicine, Baltimore, USA, 3Wolfson Unit for Endoscopy, St outer membrane protein of Chlamydia trachomatis. The amount of Mark’s Hospital, London, UK, 4Department of Surgery, St Mark’s PmpC administrated per dose was 40 lg/mice. Three immunizations Hospital, Watford Road, London, UK were performed at 2 weeks interval and the evaluation of local and systemic immune response was assessed 2 weeks after the last immu- Introduction: Colorectal cancer (CRC) is one of the major causes of nization. Conjunctival immunization with PmpC elicited higher con- cancer related mortality. Dendritic cells (DC) promote either tumour centrations of SIgA in tears in comparison to subcutaneous immunity or tolerance but their effectiveness is dependent on tissue- immunization. C57BL/6 mice showed the highest proliferation values microenvironment. Growing evidence suggests that different sites when heat-inactivated Chlamydia trachomatis was used as a stimula- within the colon may have different tumour incidence, histopatho- tor. The conjunctival application of PmpC induced skewing of logical and prognostic outcomes. There are embryological, histologi- PmpC-specific immune responses toward a Th1/Th17 profile, as cal and functional differences between the proximal and the distal determined by the stimulation of IFNc and IL-17A secretion and/or sides of the colon. However, the immunological differences between the concurrent pronounced reduction of IL-4 secretion. the compartments in CRC remain unknown; we aim to explore

differences and provide a mechanistic rational for them according to expression of miR-143 and miR-375 while miR-155, miR-223 and the activity of DC in these compartments. miR-150 were increased in this model. Probiotics were able to Methods: Phenotype, activation and migration markers of DC in restore the expression of these markers. Finally, dysbiosis and lower- mucosal and tumour biopsies from proximal versus distal colon of ing bacterial diversity was characterized in colitic mice by 454-py- CRC patients were investigated using flow cytometry and immuno- rosequencing, being restored by the treatment with each probiotic. histology. Functional studies using migration assays were performed Discussion and Conclusion: Probiotics are able to increase the to assess the effect of tumour microenvironment on the migratory diversity of microbiota as well as to alter the expression of different capacity of DC. inflammatory markers and micro-RNAs. These results support the Results: In the tumour tissues, expression of the activation markers immuno-modulatory effects of probiotics and its use in IBD. CD40, CD86 was significantly higher on DC from the proximal colon. Expression of b7 (a gut homing marker) and ILT3 (immature DC marker) did not differ between compartments. Expression of CCR7 (a lymph node migration marker) was significantly higher on 048 DC from the distal colon. In the mucosae, expression of CD40, Immunoproteasome subunit LMP7 drives the development of CD86 and CCR7 on DC was similar to that in tumour tissues. Sig- colitis-associated carcinogenesis nificantly greater migration towards CCL19 in vitro by DC from the A. Visekruna, N. Vachharajani & U. Steinhoff distal colon showed that CCR7 expression was functional. Expression Institute for Medical Microbiology, Marburg, Germany of b7 and ILT3 was significantly higher on DC from the distal colon. Colorectal cancer (CRC) is the second leading cause of cancer- Conclusion: These immunological differences suggest that tumours related death worldwide. Chronic colonic inflammation often in the proximal colon have more activated DC and should be treated progresses to CRC, whereby NF-kB-controlled expression of proin- as different entities from their distal counterparts. Right side flammatory cytokines such as TNF-a, IL-6 and IL-17A provides a tumours are less frequent, more aggressive and the survival rate is crucial functional link between augmented immune responses and lower compared with left side CRC patients. This difference could be development of colitis-associated carcinogenesis (CAC). We have due to evolution of these tumours so that some mutations in adeno- recently demonstrated the involvement of immunoproteasomes in mas in the right side of the colon enable survival of the higher the regulation of NF-kB activity. Here, we show that the immuno- immune activity/surveillance at that site. Immunological differences proteasome subunit LMP7 is essential for development of CAC. We between left and right-sided CRC and its potential impact on disease found a striking difference between wild-type (WT) and LMP7 defi- outcome may inform classification of patients with the same risk cient mice with respect to number of polyps and development of stratification for clinical trials and for the development of persona- dysplasia in the AOM/DSS murine model of colon carcinogenesis. lised treatment strategies. (Dendritic cell - Colorectal cancer) On day 76 after induction of CAC, all WT mice developed macroscopically visible adenocarcinomatous lesions, but no such lesions were detected in LMP7 deficient mice. During the course of treatment with AOM and DSS, a massive infiltration of immune cells 047 into the lamina propria of WT mice was accompanied with elevated Immuno-modulatory properties of probiotics in DSS-colitis: expression of TNF-a, IL-6, IL-17A and INF-g. On the other hand, a Impact on micro-RNA expression and bacterial diversity decreased influx of neutrophils and low production of proinflammatory cytokines were observed in LMP7 KO animals. Importantly, the F. Algieri1, A. Rodrıguez-Nogales1, J. Garrido-Mesa1, treatment with the selective LMP7 inhibitor (ONX-0914) blocked N. Garrido-Mesa1, M.P. Utrilla1, M.E. Rodrıguez-Cabeza1, the production of proinflammatory cytokines and attenuated pro- N. Chueca2, F. Garcıa2 &J.Galvez1 gression of CAC in WT mice. Thus, the immunoproteasome might 1Pharmacology, Granada, Spain, 2San Cecilio University Hospital, be a specific target for development of anti-inflammatory drugs to Granada, Spain dampen the ongoing chronic inflammation and development of Introduction: Probiotics have been reported to be useful in IBD CAC. treatment through different mechanisms, including their immunomodulatory properties and the impact in the microbiota composition. These effects can promote the down-regulation of inflammation mediators including micro-RNAs. The aim was to 049 evaluate the immunomodulatory properties of different probiotics in In vitro modelling of the effects of gluten and poly:IC on mucus- DSS model of mice colitis, emphasizing the relation between micro- secreting epithelium RNA expression and bacterial diversity. L.R. Nielsen1, S. Hansen2, T. Barington3, S. Husby4 & Methods: Male C57BL/6 mice were pretreated with E. coli Nissle H. Toft-Hansen5 1917 and L. fermentum CECT5716 at 5x108 UFC/mice/day for two 1Syddansk Universitet, Campus Vej 55, Odense M, Denmark, 2Institute weeks before DSS-induced colitis (3% solution for 6 days in the of Molecular Medicine, J.B. Winsløws Vej 21, 1., Odense C, Denmark, drinking water). Non-colitic and non-treated colitic groups were 3Department of Clinical Immunology, Sdr. Boulevard 29, Odense included as reference. Six days after colitis induction, mice were sac- University Hospital, Odense C, Denmark, 4Pediatric department, Sdr. rificed. The inflammatory status was evaluated by a disease activity boulevard 29, Odense University Hospital, Odense C, Denmark, index (DAI), qPCR of inflammatory markers and micro-RNAs, and 5Paediatric Research Unit, Sdr. boulevard 29, Odense University changes induced in microbiota populations characterized by pyrose- Hospital, Odense C, Denmark quencing. Statistical significance is set at P < 0.05. Results: Probiotics administration resulted in an intestinal anti- Celiac disease is a chronic autoimmune enteropathy of the small inflammatory effects evidenced macroscopically by lower DAI values. intestine triggered by ingestion of gluten and related prolamin pro- Biochemically, it was observed a decreased expression of pro-inflam- teins in individuals with tissue type HLA DQ2 or 8. In the lamina matory cytokines (IL-1b) and an increased expression in mucins propria of the intestinal mucosa, gluten peptides are taken up by (MUC-2 and MUC-3) and tight junction proteins. Also, it was antigen-presenting cells which induce inflammation by activation of established a micro-RNA expression profile which showed a reduced gluten-specific CD4 T cells. The integrity of the intestinal epithelial

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 27 barrier depends on a mucus layer and intercellular tight junction culturing human intestinal tissue ex vivo will confirm whether these formation. Coeliac disease is characterized by enhanced intestinal findings are applicable in a more relevant model. permeability, but the reason for this is largely unknown. In the present work, we have grown a monolayer of gut epithelial cells expressing tight junctions (caco-2) and gut epithelial cells producing mucus (HT-29). We investigated if gluten had immunotoxic 051 effect on the epithelium and if these effects were changed by the Interleukin-10 inhibits human IFNc-secreting effector T cells presence of a mucus layer. indirectly by controlling antigen-presenting cell function We added chymotrypsin-treated gluten peptides to the apical side S. Veenbergen1, M.A. van Leeuwen1, G.J. Driessen2, of the membrane of caco-2- and HT-29 cells alone, and to a co-cul- L.F. de Ruiter1, H.C. Raatgeep1, D.J. Lindenbergh-Kortleve1, ture. Trans-epithelial electric resistance (TEER) was used as a mea- L.A. van Berkel1, Y. Simons-Oosterhuis1, L. de Ridder1, sure of confluency and tight junction formation of the cells. In J.C. Escher1 & J.N. Samsom1 addition, we added poly:IC simultaneously with gluten in order to 1Department of Pediatric Gastroenterology, Rotterdam, Netherlands, mimic a viral infection to test if this had any toxic effect on the epi- 2Pediatric Infectious Disease and Immunology, Rotterdam, Netherlands thelium or a synergistic effect with gluten. Our preliminary results show no significant changes between the Chronic inflammation of the gastrointestinal tract, as seen in inflam- different treatments of the epithelium compared to bovine serum matory bowel disease, arises from abnormal reactivity of T cells to albumin-treated controls. In future experiments we will use pepsin/ commensal microbiota. Interleukin-10 (IL-10) plays a crucial role in trypsin-treated gluten peptides, and preincubate the epithelium with suppressing microbiota-specific T cell responses. However, it is poly:IC before the addition of gluten. unknown how IL-10 prevents reactivation of effector T cells in the human intestine. We have recently identified a homozygous loss-of- function mutation in the IL-10RA gene of a pediatric patient with early-onset colitis. In contrast to recently reported cases, disease 050 remission could be achieved without stem-cell transplantation, allow- Interactions between intestinal epithelial cells and ing in-depth analysis of the mechanism by which IL-10 controls Coxsackievirus B and the role of the Type III Interferons human effector T cells. Lesional intestinal tissue taken at onset of disease contained high numbers of IL-17+ and T-bet+ cells. In agree- V.M. Stone1, P. Larsson1, E. Domsgen1, U. Holmlund2, ment, IL-10 failed to control IFNc and IL-17 production by acti- E.S. Ekstrom€ 2 & M. Flodstrom-Tullberg€ 1 vated T cells derived from the IL-10RA-deficient patient in vitro. By 1Center for Infectious Medicine, Karolinska Institutet, Stockholm, coculturing CD4+ T cells and monocyte-derived dendritic cells (DC) Sweden, 2Immunology, Stockholm University, Stockholm, Sweden from the IL-10RA-deficient patient and a healthy control, we The Coxsackievirus B (CVB) family of enteroviruses (EVs) are sin- revealed that IL-10R expression on DC, and not on T cells, is impor- gle-stranded RNA viruses that have been implicated in a number of tant for controlling IFNc production, and to a lesser extent IL-17 diseases including Type 1 diabetes (T1D), mycocarditis, hepatitis and production, by effector T cells. Importantly, we demonstrated that meningitis. CVBs infect via the intestinal tract and studies suggest IL-10 plays a pivotal role in controlling the differentiation of imma- that T1D is associated with CVB infection in the gut mucosa, ture monocyte-derived DC into inflammatory DC with a disease although little is currently known about these interactions. Type III promoting phenotype. Taken together, our study demonstrates that interferons (IFNks) are important regulators in the permissiveness of IL-10 is essential to limit IFNc and IL-17 secretion by human effec- both primary human pancreatic islets and hepatocytes to CVB infec- tor T cells and reveals that IL-10 exerts its suppressive function on tion, and they have also been implicated in the control of rotavirus IFNc-secreting effector T cells mainly indirectly by controlling anti- infection in intestinal epithelial cells (IECs). However, to date, their gen-presenting cell function. role in limiting EV infection, and more specifically CVB infection in the gut is not known. Therefore, the focus of this study was to examine the interactions between CVBs and IECs and to determine whether the IFNks can regulate the permissiveness of IECs to infec- 052 tion. To examine this, we have used the two human colonic cell lines Intestinal inflammation induced by dietary cholesterol in HT-29 and CaCo2 and the common EV, CVB3. Zebrafish In this study we demonstrate that both HT-29 and CaCo2 cells F. Progatzky1, N.J. Sangha1, N. Yoshida1, M. McBrien1, express the two receptors used by CVBs to infect cells, namely CAR J. Cheung1, A. Shia2, J. Scott2, J.R. Marchesi3, J.R. Lamb1, and DAF. Moreover, we also show that both cell lines are susceptible L. Bugeon1 & M.J. Dallman1 to infection with CVB3 as confirmed by plaque assay and the moni- 1Life Sciences, Imperial College London, London, UK, 2NHLI, Imperial toring of cytopathic effects. 3 h and 6 h post infection, both cell College London, London, UK, 3School of Biosciences, Cardiff University, k lines also appear to up-regulate the expression of IFN 1 (interleu- Cardiff, UK kin-29, IL-29) and IFNk2 (IL-28a). RT-PCR analysis of the two IFNk receptor subunits, IFNk-R1 and IL-10R2, confirmed that the Inflammatory bowel disease and other chronic, often systemic, auto- IEC lines express both subunits at the mRNA level. Furthermore, inflammatory human disorders have considerately increased over the treatment of the IECs with IFNk1 and IFNk2 for 6 h and 24 h up- past decade, coincidently with a rise in the consumption of diets regulated the expression of a number of IFN stimulated genes enriched in cholesterol or saturated fatty acids. The intestinal muco- involved in antiviral defence mechanisms at both the mRNA and sal barrier first encounters dietary components, however, in vivo protein level, as confirmed by RT-PCR and Western blotting. information regarding their local and direct inflammatory effect on From this study we can conclude that the two human IEC lines, the intestinal epithelium is lacking. We found that a single exposure HT29 and CaCo2 are susceptible to infection with CVB3 and that to a high cholesterol diet (HCD) results in localized accumulation of upon infection they up-regulate the expression of the IFNks. More- myeloid cells in the intestine in both mice and zebrafish. Pharmaco- over, the IECs also enter an antiviral state after treatment with the logical interventions demonstrated that this acute myeloid cell accu- IFNks. However, whether the IFNks protect against CVB infection mulation is dependent on cholesterol uptake via NPC1L1, NFkB, remains to be established. Further studies using a novel method for NADPH oxidase and Cathepsin B, leading to capase-1 activity in

intestinal epithelial cells. Through a novel morpholino oligonucleo- obtained from an ex vivo culture of healthy human colonic mucosa tides delivery approach in zebrafish we found that inflammasome was used to condition monocyte-derived DC in an in vitro model as activation in epithelial cells is dependent on ASC. Extending the to mimic the exposure of DC to intestinal microenvironment. Con- HCD exposure to 10 days results in localised functional dysregula- ditioned--DC (C-DC) were analyzed by flow cytometry for the tion, dependent on cholesterol binding/uptake and inflammasome expression of HIV-1 receptors and activation markers, and incubated activation, and systemic pathologies. Our model reveals a novel in vitro with either R5 or X4 HIV-1 to study their susceptibility to route by which dietary cholesterol can initiate intestinal inflamma- infection. tion and suggest its involvement in long-term pathologies of the C-DC displayed an activated phenotype, a significant down-regu- intestine. Further, our model illustrates the power of the zebrafish lation of CCR5, CD4 and CX3CR1, an up-regulation of CXCR4 and system to study pathophysiological responses induced in the intesti- a moderate modulation of DC-SIGN expression compared to uncon- nal epithelium in an intact organism and aspects of mucosal immu- ditioned DC. No change in the CD103 expression was observed. nology research that have been hampered in the past. Interestingly, both R5 and X4 HIV-1 integrated their genome and replicated less efficiently in C-DC compared to unconditioned DC. Colonic supernatants contained the CCR5-binding chemokines Mip1b and MCP-1 and the CX3CR1 ligand fractalkine, whereas the 053 CXCR4 ligand SDF-1a was absent. IL-10 and IL-2, described to Intestinal mucin-dendritic cell crosstalk in gut homeostasis induce CXCR4 up-regulation on DC, were also detected. Thus, this F. Melo-Gonzalez1, T.M. Fenton1, D.J. Thornton2 & specific intestinal milieu may determine the observed phenotype. + + + M.A. Travis1 Both CX3CR1 and CD103 CD11c DCs were detected in human + 1MCCIR, University of Manchester, Grafton Street, Manchester, UK, colonic LP. Interestingly CD11c DC showed lower CCR5 and 2Wellcome Trust Centre for Cell Matrix Research, Oxford Road, higher CXCR4 expression compared to blood DC, and a similar acti- Manchester, UK vation profile, which confirmed the results obtained after intestinal conditioning. The gastrointestinal tract is lined with mucus, mainly composed of Thus, the intestinal microenvironment module the expression of the goblet cell secreted mucin MUC2. Despite this mucus layer, the HIV receptors on DCs and their capability to replicate the virus. intestine still comes into contact with the commensal microbiota Keywords: HIV-1, mucosal transmission, dendritic cells. and potential enteric pathogens. Thus, a fine regulation of the immune system in the intestine is required to maintain gut homeostasis and protect from infection. Intestinal dendritic cells (DCs) are essential for sampling luminal antigens and promoting the appropri- 055 ate immune responses to the intestinal environment, promoting tol- Isolation, culture and monitoring of Chlamydia trachomatis and erogenic responses to commensals while orchestrating effector suis using the pig model responses against pathogens. Recent evidence has suggested that T. K€aser, T. Cnudde, G. Hamonic, M. Rieder, K. Lai, A. Pasternak interactions between intestinal DCs and mucins may modulate DC & F. Meurens function and subsequent immune responses. To determine the VIDO-InterVac, 120 Veterinary Road, Saskatoon, Canada potential for mucins to regulate DC function, we treated human monocyte-derived DC with purified MUC2/Muc2 obtained from Chlamydia trachomatis infections are the most common sexually intestinal cell lines and mouse large intestine. We found that expres- transmitted bacterial disease worldwide and infection rates are sion of the pro-inflammatory chemokine interleukin 8 (IL-8) is sig- especially high in aboriginal communities. C. trachomatis infects nificantly upregulated by human DCs in the presence of MUC2/Muc2. genital tract mucosa and can cause severe health problems includ- Additionally, mucin-treated DCs are able to enhance recruitment of ing infertility and an increased risk for infections like HIV/AIDS. neutrophils in transmigration assays. Thus, in contrast to recent pub- A vaccine against C. trachomatis is currently not available but lished results suggesting potential anti-inflammatory properties of urgently needed. In order to develop such a vaccine it is impor- MUC2, we find that this mucin may induce important pro-inflamma- tant to study the immune response in a relevant animal model to tory functions in DC. Further investigation is therefore required to understand the host pathogen interactions in human chlamydial explore mucin-DC interactions during health and infection. infections. Pigs are the natural host of C. suis and infection with C. trachomatis closely resembles the infection in humans. There- fore, the pig is a relevant large animal model to study the host pathogen interactions of C. trachomatis. Additionally, the method- 054 ology for a detailed analysis of the relevant T-helper and regula- Intestinal myeloid DCs display an activated phenotype and are tory T-cell immune responses is available. The first steps in this less susceptible to HIV-1 infection compared to blood DCs establishment are the propagation, purification and titration of M. Cavarelli1, B. Fusetti1, M. Tolazzi1, K. Tsilingiri2, M. Rescigno2 chlamydia on one side and the isolation of the host cell target of & G. Scarlatti1 chlamydia, genital tract epithelial cells, on the other side. Most of 1IRRCS San Raffaele Scientific Institute, Milan, Italy, 2European the available protocols were established in the 1980s. This time Institute of Oncology, Milan, Italy gap creates two main problems: First, some of the incorporated chemicals are no longer easily available and second, most of the We recently showed that human colonic lamina propria (LP) incorporated methods are obsolete. Therefore, we updated existing + CD11c DC actively shuttle R5 HIV-1 across an intact epithelial bar- protocols with available chemicals and included state of the art + rier and transfer infection to CD4 T cells (Cavarelli, EMBO Mol- techniques like Taqman probe RT-qPCR, multi-colour confocal Med 2013). However the susceptibility of intestinal DC to HIV microscopy and flow cytometry analysis. These protocols enable infection has been poorly investigated, due to difficulties in isolating the production of a highly purified and precisely titrated stock of mucosal DC. chlamydia as well as the isolation and detailed characterization of + CD11c myeloid DC obtained from the colonic LP were further genital tract epithelial cells. These protocols will provide the basis + + characterized as CD103 and CX3CR1 and the expression of for a comprehensive analysis of the host immune response on HIV-1 receptors analyzed in comparison to blood DCs. Supernatant chlamydial infection.

056 058 Local responses to virulent and avirulent Clostridium Microbial PAMP co-stimulation initiates neutrophilic steroid perfringens in broilers resistant asthma K. Russell S. Hadebe1, G. Brown1, P. Redelinghuys1, R. Drummond1, Disease Systems, Roslin Institute Building, Midlothian, UK K. Fierens2, B. Lambrecht2, F. Brombacher3, F. Kirstein3 & S. Vautier1 The main causative agent of necrotic enteritis is C. perfringens (Cp) 1Immunity, Infection and Inflammation Programme, University of type A, which produces several toxins, including netB. Early host Aberdeen, Aberdeen, UK, 2Respiratory Diseases, University of Ghent, responses therefore an in situ duodenal loop broiler model was Laboratory of Immunoregulation and Mucosal Immuno, Ghent, developed to dissect complex host-pathogen interactions at the early Belgium, 3Immunology, University of Cape Town, Institute of stages of disease pathogenesis. Here, we investigate host responses to Infectious Disease and Molecular Medi, Cape Town, South Africa virulent, netB+, and avirulent, netB-, strains of Cp and their culture supernatants. Introduction: Asthma is a complex obstructive airway disease char- Six isolated duodenal loops were prepared surgically in anaesthe- acterised by airway hyper-reactivity to innocuous allergens. It may tised broilers. Loops within birds were infused with: control (growth be categorised as either classical eosinophilic, T helper 2 type of dis- medium), Cp culture supernatant alone or bacteria and culture ease or as one driven by neutrophils that may be associated with T supernatant from either the virulent or avirulent strain. Intestinal helper 17 cells and that is corticosteroid resistant. While the patho- segments were removed for histology and gene expression analysis at genesis of the disease is not fully understood, there is increasing evi- 0.5 or 4 h post-infusion. Expression of genes related to disease path- dence for the role of environmentally-derived pathogen-associated ogenesis (FAS, GIMAP8), immune cell activity (BLA, NBL1) and molecular patterns (PAMPs) including fungal b-(1,3)-glucans and inflammation (IL-6, IL-1â and IFN-~a) were measured. bacterial lipopolysaccharide (LPS) in inducing and exacerbating air- The main effects detected were that IL-6 expression was signifi- way inflammation. cantly down-regulated in virulent treated birds while FAS was signif- Results: We investigated the effects of these components, either icantly increased in loops containing bacteria and culture alone or in combination, in several models of pulmonary inflamma- supernatant at 4 h in comparison to the control and culture super- tion and discovered that they modified airway responses in vivo. natant alone. There were no other significant changes in pathology Notably, a combination of PAMPs drove a profound neutrophilia and heterophil infiltration between the treatments at these early time that was associated with synergistic IL-17A and RANTES production. points. The implication of the results will be discussed. Moreover, in allergic models using house dust mite, sensitisation with these agonists resulted in corticosteroid resistant airway hyper- responsiveness. Conclusion: Sensitisation with multiple microbial PAMPs may 057 therefore play an important role in the pathogenesis of steroid resis- Macrophage subsets exhibit selective endotoxin tolerance tant asthma. induced by Escherichia coli LPS K. Alshaghdali1, C. Haywood2, J. Beal3 & A. Foey3 1 School of Biomedical and Healthcare Sciences, University of Plymouth, 059 Plymouth, UK, 2Derriford Hospital, Plymouth, Plymouth, UK, 3School Microbial signals control inflammation and autoimmunity of Biomedical and Healthcare Sciences, Plymouth, UK induced by hypomorphic RAG defects Macrophages (MФs) control gut mucosal responses; facilitating tol- R. Rigoni1, V. Maina1, S. Mantero1, V. Marrella2, F. Grassi3, erance to commensal bacteria and food components, while keeping G. Pesole4, J.R. Mora5, A. Villa2 & B. Cassani2 the ability to trigger immune defences to pathogens. MФ reactions 1Humanitas Research Hospital, Via Manzoni 113, Rozzano MI, Italy, are determined by both differentiation and activation stimuli, giving 2IRGB-CNR, Via Manzoni 113, Rozzano MI, Italy, 3Institute for rise to two distinct subsets; pro-inflammatory M1- and anti-inflam- Research in Biomedicine, via Vela, Bellinzona, Switzerland, matory/regulatory M2- MФs. M2-like MФs take control in homeo- 4Universita’ di Bari, Bari, Italy, 5Massachusetts General Hospital, static environments whereas M1-like MФs predominate in Harvard Medical Sc, Boston, USA inflammatory pathologies. Suppression of MФ responses can be beneficial to either the host or pathogen. Chronic stimulation by bacte- Hypomorphic mutations in the RAG genes result in profound lymp- rial pathogen associated molecular patterns (PAMPs), such as LPS, is hopenia associated with multisystem autoimmune manifestations in well established to induce tolerance. The aim of this study was to humans and mice. The role of gut homeostasis and microbial signals investigate the susceptibility of MФ subsets to Escherichia coli K12 in the immune dysfunctions and disease pathogenesis is still debated. LPS-induced suppression. M1- and M2-like MФs were generated in The Rag2R229Q/R229Q mutant mice developed an inflammatory vitro from the THP-1 monocyte cell line by differentiation with bowel disease involving the small intestines, characterized by marked PMA and vitamin D3, respectively. MФ subsets were pre-treated infiltration of CD4+ T cells and, intriguingly, also of Treg cells, in K12-LPS prior to stimulation by bacterial PAMPs. Modulation of the lamina propria compartment. Increased expression of the gut inflammation was measured by TNFa, IL-1b, IL-6, IL-10 ELISA. homing receptors CCR9 and a4â7 on peripheral CD4+ T cells con- K12-LPS suppressed PAMP induced TNFa expression in M1 and firmed the abnormal lymphocyte trafficking to this environmental M2 but selectively suppressed IL-6 and IL-10 expression in M1 and interface. A pro-inflammatory profile, characterized by a Th1/Th17 M2 MФs. In conclusion, E. coli LPS selectively tolerises cytokine skewing, distinguished the intestinal immune responses in the production between proinflammatory M1 and regulatory M2 MФs; Rag2R229Q/R229Q mice. Remarkably, similar pattern was also evi- differential suppression exerting knock-on effects on both immuno- dent in the periphery. On the contrary, B cells were poorly present pathology and homeostasis. in the gut of mutant mice and the fecal level of IgA was obviously reduced. Interestingly, these findings correlated with augmented intestinal permeability and enhanced epithelial innate responses. Me- tagenomic analysis revealed substantial changes in the composition of the gut microbial communities in the mutant mice, with an

overall reduced bacterial biodiversity. Importantly, decreasing micro- in division, rest in quiescence or migrate/dye. This model has been bial load with antibiotic treatment significantly limited the lympho- successfully applied to analyse CD4+ T-cell priming following differ- cytic infiltration, as demonstrated by the reduction in the frequency ent mucosal routes of immunization such as vaginal or nasal immu- of peripheral CCR9 + T cells, and ameliorated both the intestinal nization and has allowed to estimate the probability of CD4+ T cells and systemic inflammation by dampening pro-inflammatory Th1/ in the draining lymph nodes to enter in division [Pettini E. et al., Th17 immune responses. Overall, these results suggest that microbial PlosOne 2013 8:e80545]. Here, a systems biology approach was factors may play a substantial role in the pathogenesis of human employed to get quantitative information on the dissemination of autoimmune disease associated with hypomorphic RAG defects. primed CD4+ T cells from draining lymph nodes (LNs) to distal lymph nodes and spleen following nasal immunization. The adoptive transfer model of transgenic OT-II ovalbumin(OVA)-specific CD4+ T cells was employed to study the antigen-specific T-cell primary 060 activation following nasal immunization. OT-II CD4+ T cells were Milieu-adjusted (physiological) oxygen concentration reduces labeled with CFSE and adoptively transferred into recipient mice, the production of IL-8 and CCL-20 upon stimulation with that were immunized with OVA and CpG ODN by the nasal route. Flagellin in CaCo-2 cells Groups of mice were sacrificed 0, 48, 60, 72, 84, 96 and 120 hours S. Klein, S. Former€ & K. Heeg following immunization, and cervical, mediastinal, iliac, and mesen- + Department of Infectious Diseases, Im Neuenheimer Feld 324, teric lymph nodes and spleen were collected. Antigen-specific CD4 Heidelberg, Germany T-cell clonal expansion was observed in draining LNs already 2.5 days following mucosal immunization, while OVA-specific prolif- Background: Intestinal epithelial cells (IECs) line the front to micro- erated T cells appeared in distal lymph nodes and spleen since bial components in the gut lumen. Their close contact to various 3.5 days following immunization. A Galton Watson multitype immune cells as well as their barrier function gives them a central branching process with emigration was successfully employed to role in maintaining immunological homeostasis in the gut. While model in vivo dissemination of proliferated T cells. The model has ambient air comprises about 20% oxygen, the partial pressure of allowed to estimate the probability of CD4+ T cells to migrate from oxygen in tissues is far below this. As many cellular and immune draining lymph nodes to distal lymph nodes and spleen. In conclu- functions are influenced by oxygen level, it may also be of impor- sion, the modeling analysis shows that the developed model is reli- tance in IECs. able and can be fruitfully exploited for predictive simulations related Methods: The colon epithelial cell line CaCo-2 was investigated to the study of primary T-cell activation and dissemination. Key- under a physiological oxygen concentration of 1%. Proliferation was words: Systems biology, T-cell dissemination, nasal immunization, measured by the incorporation of 3H-Thymidin and viability by mathematical modeling. MTT-assay. Chemokine production was measured by ELISA or intracellular by FACS, gene expression by RT-PCR and proteins were detected by Western Blot. Results: Proliferation and viability of the cells were not significantly 062 altered compared to conventionally cultured cells. While transepithe- Neonatal dendritic cell development lial electrical resistance (TEER) was initially elevated under low oxy- T.Z. Murray & A. Mowat gen, it reached comparable levels over time. When stimulated with Institute of Infection, Immunity and Inflammation, 120 University the TLR 5-ligand Flagellin, IL-8 and CCL-20 production was dimin- Avenue, Glasgow, UK ished at 1% O2. Interestingly, mRNA levels of IL-8 and CCL-20 were not significantly reduced under physiological oxygen concentration. The intestinal immune system has to discriminate between inducing Analysis of MAPK by Western Blot showed a generally higher phos- tolerance and active immunity to food proteins, commensal bacteria phorylation of p38, JNK and ERK1/2 under low oxygen, which was and pathogens. Dendritic cells (DCs) in the gut wall and draining further enhanced by stimulation with Flagellin. Inhibition of MAPK lymphoid tissues are central to these processes. We and others have did not alter the chemokine secretion. Intracellular IL-8 measure- identified four functionally distinct populations of intestinal DCs ments pointed to an alteration in chemokine transport. based on CD11b and CD103 expression. These appear to develop Conclusions: Understanding the influence of the local oxygen pres- from a common DC precursor, however little is known about how sure on the physiology of the cells could contribute to a better the local intestinal microenvironment might determine their differ- understanding of the complex immunological network in the intes- entiation and subsequent fate. We have begun to explore these tine. issues, focusing on the neonatal period when the intestinal immune system has to adapt to rapid changes in diet, anatomical structure and microbiota. All four DC subsets are present in the intestinal lamina propria 061 (LP) from birth, although there are fewer CD103+CD11b+ DCs com- Modeling T-cell dissemination upon nasal immunisation using a pared with the adult. However during the first three weeks of life, systems biology approach there is a progressive increase in the proportion of CD103+CD11b+ G. Prota1, A. Boianelli2, E. Pettini1, G. Pozzi1, A. Vicino2 & DCs, and these become the majority population around weaning, D. Medaglini1 resembling the composition of the DC compartment in the adult. 1L.A.M.M.B., Dipartimento di Biotecnologie Mediche, Universitadi Few migratory or resident DCs are found within the draining Siena, Siena, Italy, 2Dipartimento di Ingegneria dell’Informazione, mesenteric lymph node (MLN) at birth, but both populations CSC, Universita di Siena, Siena, Italy increase gradually in number thereafter. Consistent with their migratory origin from the LP, the numbers of CD103+CD11b+ and The application of systems biology in vaccinology has recently been CD103+CD11b- DCs show delayed appearance compared with the proposed as powerful tool to characterize immune responses to vac- mucosa. However by three weeks of age, the proportions of DC sub- cination and to predict vaccine immunogenicity and efficacy. Our sets in the MLN are comparable to those observed in adults. group has recently employed a branching process to model in vivo Together these data support the idea that intestinal environmental + CD4 T-cell priming and estimate the probabilities of a cell to enter factors drive differentiation of DC subsets in distinct, age-dependent

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 31 ways. Specifically CD103+CD11b+ DCs, unique to the intestine, are on eosinophils is one sensitive surrogate biomarker for activation, highly dependent on changes within the local environment. Identifi- accumulation and recruitment to inflammatory sites. cation of these factors will aid understanding of how DCs induce Method: We sought to optimize the assay in terms of sample post- tolerance against commensal bacteria and food proteins. collection stability, staining duration and gating strategy for clinical implementation purposes. The criteria for this evaluation are based on the percent and CD11b expression in median fluorescent intensity of eosinophil. The gating strategy in identifying eosinophils are aug- 063 mented by their auto-fluorescent properties and CD11b expression Novel markers to evaluate immunomodulatory effects of instead of the classical definition characterized by CD9hi and CD16dim antibiotics in DSS-colitis: micro-RNA expression and microbiota phenotype which shows overlap with our new method. Basically, the populations eosinophil events, derived from total granulocytes, are identified on J. Garrido-Mesa1, A. Rodriguez-Nogales1, F. Algieri1, the bi-variate plot with FITC, an empty channel versus side scatter N. Garrido-Mesa1, M.P. Utrilla1, M.E. Rodriguez-Cabezas1, parameters, as they are most distinctive on the lower spectrum of < F. Garcia2, N. Chueca2 & J. Galvez1 wavelength. We chose an acceptance range for the criteria to be 1Pharmacology, Avenida del Conocimiento s/n, Granada, Spain, 20%CV comparing to those parameters at 0, 4, 6, 24 and 48-hour. 2Universitary hospital San Cecilio, IBIG, Granada, Spain Results: Among five different anti-coagulant collection tubes, K2EDTA and 3.2% Sodium Citrate anti-coagulants shown to have at Introduction: Minocycline and doxycycline exert immunomodula- least 24-hour stability window, which is important to further this tory effects that could be beneficial in IBD. Micro-RNAs have been assay for clinical diagnostics. As a result, the optimized flow panel recently reported to play a key role in intestinal homeostasis that design in conjunction with this new gating strategy and the extended can be influenced by microbiota populations. The aim of the study sample stability give the assay more flexibility, efficiency and an was to evaluate the effect of these antibiotics in the DSS-colitis additional clinical tool aiding in allergic disorders evaluation as well model in mice, characterizing the modifications induced in the as its utilization in clinical trials. micro-RNA expression and bacterial diversity. Methods: Male C57BL/6J mice were assigned into non-colitic and DSS-colitic groups. Colitis was induced by dextran sodium sulfate (DSS) in the drinking water (3%) for 6 days. Once the colitis pro- 065 cess was established, colitic mice were divided in three groups: DSS- Oral antigen modulates primary systemic KLH-specific T- and control (without treatment), MNC (receiving minocycline 50 mg/kg/ B-cell responses in humans day) and DXC (receiving doxycycline 10 mg/kg/day). After six days T. Meyer1, B. Boortz1, C. Giesecke2, J. Maul1, J. Preiß1, of treatment, all mice were sacrified. The inflammatory status was T. Doerner2 & R. Ullrich1 evaluated by a disease activity index (DAI), qPCR of inflammatory 1Gastroenterology, Berlin, Germany, 2Rheumatology, Berlin, Germany markers, including micro-RNAs. Also, changes in microbiota populations were characterized by pyrosequencing. Oral tolerance is an antigen-specific process and refers to specific Results: According to the DAI values, minocycline and doxycycline non-reactivity of the immune system against an antigen that has treatment improved the recovery of colitic mice, ameliorating some been fed to the organism and thus comes into contact with the of the inflammatory markers, including IL-1b, IL-17, TGFb, MMP- immune system via the mucosa of the gastrointestinal tract. Espe- 2, MUC-2, MUC-3, ZO-1 and occludin. A micro-RNA expression cially in humans, the underlying mechanisms are poorly understood. profile was established for this model of colitis showing an increased In our clinical study we administered 50 mg KLH/d for 10 days expression of miR-155, miR-223 and miR-150 while miR-143 and to healthy volunteers of the oral (n = 8) but not the control group miR-375 were decreased. Both antibiotics partially restored the (n = 8). Afterwards both groups were subcutaneously immunised expression of some of these markers. Pyrosequence characterization with KLH. A DTH skin reaction was then provoked by an intrader- of microbiota showed that minocycline and doxycycine treatments mal KLH injection. Follow-up investigations were done after increased the bacterial diversity, reverting the dysbiosis produced by 6 months. During the study PBMC were isolated at defined time DSS-colitis. points and KLH-specific T and B cells were analysed. The propor- Discussion and Conclusion: Minocycline and doxycycline are able tions of cytokine-producing KLH-specific CD4 T cells and KLH- to modify the expression of different inflammatory markers and mi- induced proliferation of CD4 T cells were analysed after in-vitro res- croRNAs, as well as to increase the intestinal bacterial diversity. timulation. KLH-specific B cells were identified by fluorescent- These observations confirm the combined contribution of antibiotic labeled KLH. KLH-specific serum IgG, IgA and IgM as well as IgG and immunomodulatory properties ascribed to these compounds subclasses were measured by ELISA. that could be of great interest to control the complex pathogenesis Only 2/8 volunteers of the oral group but 7/8 volunteers of the of IBD. control group developed a subsequent skin reaction. KLH-specific proliferation and frequencies of KLH-specific CD154+ CD4+ T cells producing IFN-gamma, IL-2 and TNF-alpha were higher in volunteers with DTH compared to those without a DTH. Oral KLH 064 primed rather than suppressed KLH-specific B cells. Primary KLH- Optimization of a flow cytometric peripheral eosinophil assay specific plasma cells were not predominantly IgM+ but commonly aiding in diagnosis and monitoring of allergic disorders expressed IgA. M. Tseng, L. Austin, M. Ryherd, R. Limgala, M. Plassmeyer, Tolerance induction in humans by oral antigen was confirmed M. Brown, R. Gupta & O. Alpan only in part. In particular, KLH-specific proliferation was not abol- Amerimmune, 11212 Waples Mill Road Suite 100, Fairfax, VA, USA ished after oral KLH. Introduction: Eosinophilic inflammation is an important component of the pathology seen in allergic disorders such as eosinophilic gastrointestinal disorders and asthma. Expression of an integrin, CD11b

066 els of inhibitory citokynes such as IL-10. The aim of this study is to Oral tolerance failure upon neonatal gut colonization with correlate the cytokines production to the pathology caused by infec- Escherichia coli producing the genotoxin colibactin tion. TS strain have higher weakness, apathy, prostration and mortality due to hepatosplenomegaly more intense due to the larger size T. Secher1, D. Payros2, C. Brehin3, M. Boury2, C. Watrin2, of hepatic granulomas and extensive fibrosis of these granulomas. M. Gillet4, I. Bernhard-Cadenat1, S. Menard4, V. Theodorou4, Both strains produced IFN-~a, but TS produced IL4 and IL-10 in a A. Saoudi1, M. Olier4 & E. Oswald1 larger quantity, however IL-10 was not able to regulate the growth 1INSERM-U1043, Toulouse, France, 2INRA-USC1360, Toulouse, of hepatic granulomas exacerbated this lineage. High levels of IL-4 France, 3CHU Purpan, Toulouse, France, 4INRA-UMR1331, Toulouse, in TS strain are consistent with the exacerbation of granulomas, France since IL-4, as well as IL-13, induces collagen synthesis and is related Introduction: The commensal bacterium E. coli belongs to the pio- to the development of fibrosis in schistosomal granuloma. Additional neer microflora colonizing the mammalian gut immediately after studies are needed to confirm our proposals and to understand the birth. E. coli can be differentiated in four major phylogenetic groups mechanisms underlying the difference in immune response of these (A, B1, B2 and D). Recent studies showed that B2 group, including strains in the schistosoma-host relationship. strains producing the genotoxin colibactin, is over-represented while its prevalence is increasing among E. coli strains persisting in the microbiota of humans from developed countries, including infants. In parallel, recent evidences demonstrate that inflammatory-related 068 diseases are on the increase in developed countries. Neonatal intesti- Oral tolerance susceptibility is related to higher reduction of B nal microbiota, through the modulation of metabolism and immu- lymphocytes subpopulations in schistosomiasis. nity may have long-term consequences on host susceptibility to such D. Tavares1, A.C. da Silva1 & A. Xavier2 diseases. 1Departamento de Genetica, Rua S~ao Francisco Xavier, 524 - PHLC, Aims and Methods: Using an original rodent model of neonatal gut Lab. 200C, Rio de Janeiro, Brazil, 2Departamento de Imunobiologia, colonization allowing the natural vertical transmission and persis- Universidade Federal Fluminense (UFF) tence of E. coli strains from mother to the offspring we analyzed the effects of genotoxic commensal B2 E. coli on the host oral tolerance The Schistosoma mansoni infection was studied in two strains of to intestinal antigen. Pregnant rats were fed with a human commen- mice genetically selected for extreme phenotypes of susceptibility sal B2 E. coli, an isogenic non-genotoxic mutant, or an isogenic (TS) and resistance (TR) to oral tolerance. TR strain present good genotoxic complemented mutant. After weaning, rats were submitted inflammatory responses and a non-tolerogenic profile while TS are to an OVA oral tolerance and delayed-type hypersensitivity (DTH) non-inflammatory but high-tolerogenic, with high percentages of + + + protocol. CD4 CD25 Foxp3 T regulatory cells, and able to produce high lev- Results: We report here that colibactin expression increased intesti- els of inhibitory citokynes such as IL-10. The aim of this study is to nal permeability and enhanced intestinal translocation of B2 E. coli correlate the cytokines production to the pathology caused by infec- in neonates. At adulthood, we observed impaired intestinal barrier tion. TS strain have higher weakness, apathy, prostration and mor- together with an increase of the permeability through Peyer’s patches tality due to hepatosplenomegaly more intense due to the larger size correlated with enhanced IFN~a levels. This alteration of the gut bar- of hepatic granulomas and extensive fibrosis of these granulomas. rier homeostasis exacerbated the mucosal and systemic immune Both strains produced IFN-~a, but TS produced IL4 and IL-10 in a responses against a luminal antigen through an experimental model larger quantity, however IL-10 was not able to regulate the growth of OVA-driven oral tolerance. Animals early colonized by colibactin- of hepatic granulomas exacerbated this lineage. High levels of IL-4 producing E. coli exhibited enhanced OVA-specific response, which in TS strain are consistent with the exacerbation of granulomas, was correlated with a defect in Tregs population. Submitted to a since IL-4, as well as IL-13, induces collagen synthesis and is related mucosal DTH reaction, colibactin-producing E. coli colonized ani- to the development of fibrosis in schistosomal granuloma. Additional mals displayed exacerbated signs of inflammation which was also studies are needed to confirm our proposals and to understand the correlated to a decreased in Tregs population. mechanisms underlying the difference in immune response of these Conclusion: The neonatal gut colonization by E. coli producing the strains in the schistosoma-host relationship. genotoxin colibactin enhances intestinal translocation and subsequently alters oral tolerance. Thus, through the production of colibactin, early gut colonization by commensal E. coli may represent a 070 risk factor for the development of immune-mediated diseases. Oral tolerance susceptibility is related to ultrastructural alterations in adult female and male Schistosoma mansoni A. Xavier1, D. Tavares2, E. Guimar~aes3, A.C. da Silva2, M. Neto2 & 067 A. Henrique3 Oral tolerance susceptibility is related to exacerbated 1Departamento de Imunobiologia, Niteroi, Brazil, 2Universidade do granuloma development in schistosomiasis. Estado do Rio de Janeiro (UERJ), Brazil, 3Fiocruz, Brazil 1 1 2 D. Tavares , A.C. da Silva & A. Xavier The Schistosoma mansoni infection was studied in two strains of 1 ~ Departamento de Genetica, Rua Sao Francisco Xavier, 524 - PHLC, mice genetically selected for extreme phenotypes of susceptibility 2 Lab. Rio de Janeiro, Brazil, Departamento de Imunobiologia, (TS) and resistance (TR) to oral tolerance. The objective was to ana- Universidade Ferderal Fluminense (UFF), Brazil lyze by Transmission Electron Microscopy the influence of the host The Schistosoma mansoni infection was studied in two strains of immune regulatory profile on the worm morphology. Parasites mice genetically selected for extreme phenotypes of susceptibility recovered from TR mice showed no morphological changes. How- (TS) and resistance (TR) to oral tolerance. TR strain present good ever, specimens collected from TS mice, exhibited tubercle swelling inflammatory responses and a non-tolerogenic profile while TS are with blunted and shortened spines in lower density. These tegument non-inflammatory but high-tolerogenic, with high percentages of alterations were similar to those described with artemether or pra- CD4+CD25+Foxp3+ T regulatory cells, and able to produce high lev- ziquantel treatment, supporting observations that the host immune

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 33 system influences the tegument development and function of worms lamina propria (LP) and spleen. Frequency of these cell subsets harbored in non anti-helminthic treated TS mice. The ileum oogram increased in gut mucosa while it decreased in spleen, suggesting a from TS mice showed a higher percentage of dead eggs and a lower domiciliation defect after perinatal BPA treatment. Concomitantly, a percentage of immature eggs than TR mice, but had similar quanti- decrease of regulatory and activated T cells in LP and mesenteric ties of collected eggs. This suggests that in TS mice the alterations in lymph node was observed while these sub-populations were adult worm tegument prevented egg development, as a consequence increased at splenic level. Interestingly, an alteration in the frequency of decreased glycogen granules and extensive lysis of internal struc- of innate lymphoid cells ILC3 producing IL-17 and IL-22 occurred tures, but not egg production reduction. These results corroborate in LP, associated with a decrease of aryl hydrocarbon receptor (AhR) our previous Scanning Electron Microscopy study indicating the expression, known to drive the development of gut ILC22. Our influence of the host immune regulatory profile on the development results demonstrated that perinatal exposure to BPA weakens protec- and function of the worm reproductive system and tegument. tive and regulatory immune functions of IEC associated with an alteration of DC and T cells populations and ILC3 development. These disturbances in the progeny might impair mucosal tolerogenic responses, and favored inflammatory systemic immune responses, 071 thus increasing susceptibility to food adverse reactions. Pathogenesis of pouchitis--analysis of adherent junction proteins

1 1 2 2 F. Lasitschka , M. Tossing , M. Kadmon & C. Leowardi 073 1Pathologisches Institut, Im Neuenheimer Feld 224, Heidelberg, Post-natal mucus barrier formation in the small intestine and Germany, 2Chirurgische Universit€atsklinik, Im Neuenheimer Feld 110, distal colon Heidelberg, Germany G.M.H. Birchenough, M.E.V. Johansson & G.C. Hansson Introduction: Pouchitis, which is defined as the inflammation of the Medicinal Biochemistry and Cell Biology, Medicinaregatan 9A, ileum mucosa in the pouch, is one of the major complications after Gothenburg, Sweden restorative proctocolectomy and Ileoanal Pouchimplantation (IAP) in ulcerative colitis (UC) patients. The severity of symptoms varies Intestinal mucus is secreted by epithelial goblet cells (GC) and is and quality of patient’s life are impaired. There are indications that composed of a limited number of proteins including the gel-forming the pathogenic mechanisms of the underlying disease, i.e. ulcerative mucin Muc2. The mucus forms a barrier that restricts microbiota colitis are associated with a disturbed response to bacterial antigens access to the epithelium by region-specific mechanisms. In the colon in the pathogenesis of pouchitis. An important mechanism in this this is dependent on stacked, tissue adherent multimeric sheets of case represents a failure in the intestinal barrier, which is formed by Muc2 which form an impenetrable filter between bacteria and the antimicrobial peptides, the mucus itself, tight and adherent junctions colonic tissue. In the small intestine the Muc2 network is non-adher- between the intestinal epithelial cells and the mucosal immune sys- ent and penetrable, but forms a matrix containing antimicrobial tem. peptides. Although developmental aspects of GC biology and mucin Methods: Screening of adherent junction proteins by RTqPCR pro- composition/expression have been partially explored, surprisingly lit- filer arrays in a defined collective of pouchitis samples. Validation of tle is known regarding the timing of functional mucus barrier for- target genes by qPCR in an independent collective. Analysis of target mation and the factors which influence this process. This is of antigens by immunohistochemistry. Results: Severe pouchitis in interest considering the susceptibility of neonates to certain intestinal patients with ulcerative colitis shows a significant dysregulation of pathogens and necrotizing enterocolitis. various adherent junctionproteins compared to the pouch samples Initial mucus barrier formation was characterised in the small of FAP patients. intestine and distal colon by ex-vivo analysis of rat tissue using a Conclusion: Pouchitis in ulcerative colitis patients is characterized horizontal Ussing chamber-like system that allows quantification of by a dysregulation of adherent junction proteins compared to FAP mucus thickness, penetrability and growth rate. Immunohistochemi- patients and may contribute to a fundamental difference between the cal staining of methanol-Carnoy fixed tissue sections with antibodies two disease entities. against both the apoprotein and mature forms of Muc2 allowed in-vivo assessment of mucus barrier formation, Muc2 synthesis/secretion and GC distribution. Tissue obtained from both conventionally- raised (ConvR) and germ-free (GF) mice was used to assess the 072 potential role of bacterial colonisation in barrier formation. Analysis Perinatal exposure to bisphenol A (BPA) leads to inappropriate of the microbiota was undertaken using qPCR targeting the bacterial innate and adaptative immune responses in intestinal and 16S gene with universal and taxon-specific primers. systemic compartments at adulthood Although GCs expressed and secreted Muc2 in-utero, generation L. Guzylack-Piriou, S. Menard, C. Cartier, C. Lencina, E. Gaultier of a functional mucus layer in both the small intestine and the distal & E. Houdeau colon occurred post-natally. Colonic barrier formation was charac- INRA, 180 chemin de tournefeuille, Toulouse, France terised by rapid alterations in mucus properties and GC distribution preceding a more steady increase in the rate of mucus processing. Oral route is the major route of exposure to the food contaminant Slower barrier formation was observed in the small intestine, charac- BPA, and intestine the first organ exposed. In rodent models, BPA terised by the gradual release of stored Muc2 from villus GCs. Con- has demonstrated its ability to interfere with the gut immune system, vR barrier development was strongly correlated with increased particularly when perinatal exposure occurred, and predisposing to bacterial numbers and absent in GF animals providing a strong caus- food intolerance at adulthood. In this study, we report that adult ative link between colonization and initial barrier formation. mice perinatally exposed to a low dose of BPA [5 µg/kg BW/day] showed reduced retinoic acid (RA) production by intestinal epithelial cells (IEC). A decrease in lysozyme secretion, and total IgA and IgG production in feces was also observed. These alterations were associated with a defect in dendritic cells maturation (DC) from

074 lacking the AvrA virulence gene we observed distinct differences in the Protease-activated receptor 2 is required for inflammation of the pattern of phosphorylation within the ErbB signaling pathway. This small intestine triggered by T. gondii in C57BL/6 mice included not only the ceca, the natural site of infection, but also the adjoining jejunum, indicating that the AvrA mutant Salmonella is able C. Bonnart1, G. Feuillet1, V. Vasseur1, N. Cenac1, B. Gilmore2 & to alter signaling at alternative sites along the gastro-intestinal tract. N. Blanchard1 1Inserm U1043 CPTP, place Baylac, Toulouse, France, 2School of Pharmacy, 97 Lisburn Road, Belfast, UK Toxoplasma gondii (T.g.) is a widespread parasite present on all con- 076 tinents. Infection by T.g. persists in one third of the human popula- Regulation of Intestinal Immune Responses by the Atypical tion and can cause severe neurological and ocular pathologies called Chemokine Receptor ACKR4 toxoplasmosis. T.g. naturally enters the organism via the oral route C. Thomson, A. Mowat & R. Nibbs and crosses the small intestinal barrier to reach internal organs of the Institute of Infection, Immunity and Inflammation, University of body. Interestingly, in several mammals, small intestine inflammation Glasgow, UK is a pathological feature associated with T.g. infection, which is highly reminiscent of the Crohn’s disease (CD) pathology. Previous studies Whether they are aimed at conferring protective immunity or toler- have highlighted the contribution of proteases, protease-activated ance, immune responses in the small intestine are fundamentally receptors (PAR) and their signaling pathways, as inflammatory modu- dependent on the action of chemokines and their cognate receptors. + lators in the gut. Using the gut inflammation model triggered by T.g. For example, CCL19 and CCL21 facilitate the migration of CCR7 infection in C57BL/6 mice, we show that the development of intesti- mucosal dendritic cells from the lamina propria to the mesenteric + nal inflammation requires active PAR2 signaling. Following gavage lymph nodes (MLN), whilst CCL25 recruits CCR9 gut-homing leu- with T.g. cysts, PAR2-deficient animals exhibited milder inflamma- kocytes into the lamina propria from the circulation. Interestingly, tion, characterized by lower microscopic and macroscopic inflamma- CCL19, CCL21 and CCL25 also bind to ACKR4 (CCRL1), an atypi- tion scores and lower up-regulation of pro-inflammatory mediators cal chemokine receptor that is expressed in both the intestine and IL-6 and KC. In addition, a number of pro-inflammatory polyunsat- the MLN. In vitro studies show that, rather than inducing cell turated fatty acid metabolites were increased in infected wild-type ani- migration, ACKR4 binds and internalises its ligands, targeting them mals but not in PAR2-deficient animals. Interestingly, production of for intracellular degradation. ACKR4 is therefore proposed to regu- IFNc and TNFa, two key effector cytokines in this pathology, was not late chemokine receptor dependent migration by scavenging extracel- affected by PAR2 deletion. Finally, using innovative protease activity- lular chemokines. However, the expression and function of ACKR4 based probes, our data revealed the existence of a clear disequilibrium in vivo, particularly in the intestinal immune system, has not been in active serine proteases between inflamed and normal gut tissues. investigated in any great depth. Here, using ACKR4-eGFP reporter Identification of these dysregulated proteases is underway to investi- mice and ACKR4-deficient mice, we have identified which cell types gate their role in the PAR2 signaling cascade leading to gut inflamma- express ACKR4 in the lamina propria and gut-associated lymphoid tion in the small intestine. This study may pave the way towards the tissues. Furthermore, we have examined the impact of ACKR4 defi- discovery and modulation of new key proteases, potentially relevant ciency on immune responses in the small intestine. These studies for the treatment of intestinal inflammation disorders. aim to establish the role of ACKR4 in intestinal immunity, and dissect the complexity of chemokine networks in the small intestine.

075 Querying the intestinal host-pathogen response to salmonella 077 infection using high-throughput kinomics Relationship between Th17 and Treg in nasopharynx-associated lymphoid tissue and their association with age and R. Arsenault, C. Swaggerty, K. Genovese, H. He & M. Kogut pneumococcal carriage in humans United States Department of Agriculture, College Station, USA A. Mubarak1, V. Casey2, N. Upile2, C. Xie2, R. Sharma2, H. Beer3, Enteric pathogens have a profound impact of the functioning of the M. McCormick3, M. Ahmed1, P. McNamara4 & Q. Zhang1 gastrointestinal tract. Serovars of Salmonella elicit a strong pro-inflam- 1Clinical Infection and Immunology, Liverpool, UK, 2ENT Department, matory response in species such as humans and bovine. However, in Alder Hey Children’s Hospital, Liverpool, UK, 3ENT Department, species such as mice and chickens a significant immune response is Royal Liverpool University Hospital, Liverpool, UK, 4Department of not observed and the bacterium appears to be well tolerated. Salmo- Women’s and Children’s Health, University of Liverpool, Liverpool, UK nella only elicit disease in mice through the use of antibiotic pre-treatment and in chickens by infecting the animals very early post-hatch. Streptococcus pneumoniae is a leading cause of respiratory tract This differential response is of significant interest as it may elucidate infection in humans. S. pneumoniae colonizes human nasopharyngeal mechanisms of Salmonella pathogenesis, host response and microbiota mucosa, and carriage is common in young children that may account tolerance. Our group has considered two serovars of Salmonella, Ty- for the high incidence of pneumococcal disease in this age group. phimurium and Enteritidis, and the host-pathogen interactions in the Pneumococcal carriage rate in humans decreases with age due to nat- chicken gut. We have performed this analysis at the kinome level; the ural immunity. However, the immunological factors responsible for differentially active kinases within the gut tissue. We compared the clearance of pneumococcal carriage remain undefined. Recent infected and non-infected animals for host response. Our observations studies in mice suggest Th17 is important in host clearance of S. point to the induction of a response at the interface of immunity and pneumonia. We have studied the relationship between numbers of + metabolism. The key cellular signaling intermediates that have mucosal Th17 and T regulatory cells (Foxp3 Treg) in human naso- appeared in our analyses include mTOR, GSK3B, MAPKs, AKT, and pharynx-associated lymphoid tissue (NALT) and age and their associ- AMPK. We have observed that approximately 4 days post-infection ation with pneumococcal carriage in children and adults. Numbers of the response switches from one that is pro-inflammatory to one that Th17 and Treg in adenotonsillar tissue were significantly higher than suppresses immune response generating a tolerance of infection. In in peripheral blood (P < 0.01) in both children and adults. There was addition, in a study involving the Salmonella Typhimurium mutant a negative correlation between numbers of Th17 and Treg in adeno-

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 35 tonsillar tissue (r = À0.52, P < 0.01). Numbers of adenotonsillar 079 Th17 were shown to correlate with patients age (r = 0.62, P < 0.01), T cell receptor repertoire in refractory celiac disease as revealed whereas numbers of Treg were inversely correlated with patients age by next generation sequencing (r = À0.50, P < 0.01). Furthermore, there was a positive correlation M. Schumann1, J.-M. Ritter2, K. Zimmermann2, B. Siegmund1, between the Th17/Treg ratio in NALT and age of patients. Also, the A. Rosenwald3, S. Daum1 & M. Hummel2 Th17/Treg ratio was significantly higher in pneumococcal carriage 1Department of Gastroenterology, Charite, Hindenburgdamm 30, negative than positive children (P < 0.05). Our findings suggest that Berlin, Germany, 2Institute of Molecular Pathology, Charite, Berlin, the balance/ratio of mucosal Th17 and Treg in nasopharynx is a criti- Germany, 3Institute of Pathology, University of Wurzburg,€ Wurzburg,€ cal determinant of pneumococcal clearance/carriage, and support Germany efforts to promote mucosal Th17 in vaccination strategy against pneumococcal infection in humans. Introduction: Refractory celiac disease (RCD) is a Marsh III enteropathy with accompanying malabsorption in spite of stringent gluten-free diet (GFD) for >1 year. Contrary to type-I RCD, a clonal T cell population builds up in type-II RCD and develops further to 078 overt T cell lymphoma in a high percentage of cases. In most institu- Retinoic acid modulates the early expansion and differentiation tions, diagnostics to differentiate the two subtypes is based on Gene- + of CD4 T cells during the development of intestinal Scan technology determining fragment sizes of PCR-amplified T cell inflammation receptor (TCR) DNA sequences and immunohistology. However, A. Rivollier1, L. Pool2, U. Frising2 & W. Agace1 phenomena as “oligoclonality” and “pseudoclonality” are frequently 1Danish Technical University, Veterinary Institute, Frederiksberg C., found in GeneScans and add to a specificity problem. Moreover, the Denmark, 2Lund University, Experimental Medical Sciences, Lund, sequences of the variable regions of the involved TCRs are unknown. Sweden Aims: To shed further light on TCR biology in RCD and to do a proof-of-concept study for diagnostic testing. Epidemiological studies of vitamin A-deficient populations have Methods: Determination of the T cell repertoire of duodenal mucosa illustrated the importance of vitamin A and its metabolite all-trans by multiplex PCR of TCRb-CDR3 regions. Next generation sequenc- retinoic acid (RA) in mucosal immune responses. In support of that ing (NGS, Illumina HiSeq 2000) of amplicons in controls, active cel- finding, we and others have shown that RA produced by intestinal iacs (ACD), celiacs with GFD, unclassified Marsh I cases, RCD type- dendritic cells is required for the generation of gut-tropic T cells and I and RCD type-II. IgA-secreting plasma cells. Evidences from the literature suggest Results: On average, 106 sequences per sample were analyzed, yield- antagonistic roles for RA in intestinal T cell biology. In the homeo- ing ~1000 TCR rearrangements. Clonotypes were defined as identical static intestine RA plays anti-inflammatory functions, sustaining the sequences of CDR3 amplicons. Averaging all RCD type-II, the most + development of foxp3 regulatory T cells, while restraining Th17 frequent clonotype was 52.5% of all clonotypes per sample, which effector T cell differentiation. In contrast, the role of RA in the con- was significantly higher than in controls (16.7%; P < 0.01) or RCD trol of intestinal CD4 T cell responses under infectious or inflamma- type-I (17.3%; P < 0.001, cut-off>0.5%). ACD revealed a non-signif- tory conditions remains to be more clearly defined. icant tendency towards a higher frequency. Interestingly, patients + RA has been shown to control the CD4 T cell compartment by with high clonotype frequencies developed significantly more often a signaling through the RA receptor RARa. We therefore took advan- T cell-lymphoma. The technique was validated by identifying the tage of mice expressing a dominant negative form of the RARa, spe- same top clonotypes in various individuals receiving two examina- cifically in the CD4 T cell compartment (RARdn CD4Cre+ mice), in tions months apart. When searches for alignments with previously + order to conditionally ablate RA signaling in CD4 T cells during published CDR3 sequences were done, a significant portion of these the development of intestinal inflammation in the T cell transfer sequences was found in our celiac samples, however not within the colitis model. high frequency clonotypes (HFC). Moreover, HFC´s of individual Histological, flow cytometry and real time PCR analyses indicate RCD II patients did not reveal cross-homology, arguing against spe- + that RA signaling negatively modulates the early expansion of CD4 cific TCR motifs in RCD II pathophysiology. T cells following their transfer into RAG-/- mice, but also their dif- Conclusion: TCRb-NGS-analysis reproducibly detects monoclonal T ferentiation into IL-17-producing cells in a RORct-dependent man- cell populations in RCD-II. The TCR-CDR3-HFCS in RCD-II are + ner. Indeed RA signaling-deficient CD4 T cells selectively not previously described, gliadin-associated TCR sequences, which accumulate and showed increased capacities to differentiate into IL- might explain their potential to expand gliadin-independently. 17+, IL-17+IFNc+ and foxp3+ cells, but reduced capacities to differentiate into IFNc+ cells and were less potent at inducing intestinal inflammation. RA signaling-deficient CD4+ T cells were also unable to differentiate into cytotoxic T lymphocyte (CTL)-like cells within 080 the intraepithelial lymphocyte compartment of the colon. We are T cell specificity towards native versus deamidatede gluten currently deciphering the mechanisms of these effects of RA on epitopes in celiac disease CD4+ T cells. K.S. Rasmussen1, A. Staal1, C. Nielsen2, T. Barington2, S. Husby3 & H. Toft-Hansen3 1University of Southern Denmark, Denmark, 2Clinical Immunology, Odense University Hospital, Denmark, 3H. C. Andersen Children’s Hospital, Odense University Hospital, Denmark Background: Celiac disease (CD) is a chronic, inflammatory disease of the small intestine, which occurs in genetically predisposed individuals after ingestion of gluten from wheat, barley and rye. The gluten-specific CD4+ T cells play a crucial role in the pathogenesis of CD, where T cell receptors (TCR) recognize gluten epitopes displayed by HLA-DQ2/8 molecules. Deamidation of gluten epitopes by

tissue transglutaminase 2 is known to enhance the binding capacity Conclusion: Our findings uncover a crucial role of IL-9 producing to HLA-DQ2/8. TCRs contain highly variable regions called comple- effector T cells in murine ulcerative colitis and colitis-associated neo- mentarity-determining regions (CDR). In this study, we investigated plasias. The presence of PU.1+ CD4+ -expressing cells in inflamed whether different mucosal gluten-specific CD4+ T cells from CD and tumorigenic tissue suggests that these cells belong to the recently patients recognize native and/or deamidated peptides. described Th9 T cell subset. Methods: We established five gluten-reactive T cell lines (TCLs) from duodenal biopsies obtained from four children and one adult with CD. The reactivity of the TCLs was tested in proliferation assays against a panel of four deamidated and three native gluten 082 epitopes. A pair of one deamidated and one native epitope that elic- The anatomical origins of migratory dendritic cells in the ited the strongest T cell proliferation in all TCLs was used in nested- intestine PCR-based CDR3 diversity determination of one TCL. S. Houston1, V. Cerovic1, O. Schulz2, O. Pabst2 & S. Milling1 Results: We found that all TCLs proliferated after stimulation with 1Institute of Infection, Immunity and Inflammation, GBRC, 120 both native and deamidated peptide. The response to deamidated Univesity Place, Glasgow, UK, 2Institute of Immunology, Carl- peptide was significantly stronger. The CDR3 length distributions of Neuberg-Str. 1, Building I11, Hannover, Germany responding cells in one TCL displayed overall three different patterns according to the CDR3 lengths. Dendritic cells (DCs) are fundamental in controlling immune Conclusion: We confirmed that the deamidation of gluten epitopes responses in the small intestine (SI) and colon; they migrate to the enhance the T cell proliferation, but that native peptides also induce mesenteric lymph node (MLN) and prime gut-tropic effector or reg- a response, albeit lower. We cannot yet make conclusions based on ulatory T cells. The aim of this work was to determine whether the our preliminary results from CDR3 diversity determination of one DCs that migrate to the MLN from the intestine originate in the TCL. lamina propria (LP), or from the Peyer’s patches (PP). First, the anatomical organisation of the lymphatic vessels draining to the MLN from the SI and colon was investigated. Once this had been established, the migration of DCs from the LP and PPs to the MLN 081 was examined. TH9 cells drive IL-9 mediated colitis-associated colorectal Here, I demonstrate the anatomical segregation of DCs that cancer (CAC) migrate to the MLN from the SI and colon. After SI photoconver- sion in fluorescent transgenic Kaede mice, converted DCs were K. Gerlach1, H.-A. Lehr2, A. McKenzie3, M.F. Neurath1 & found only in the SI-draining MLN. Likewise following photocon- B. Weigmann1 version of the colon in Kaede mice, converted DCs were detected 1University Erlangen-Nuremberg, Medical Clinic 1, Erlangen, only in the colonic-draining MLN. To examine the functional conse- Germany, 2Institute of Pathology, Campus Bodensee, Friedrichshafen, quence of this anatomical compartmentalisation of lymphatic drain- Germany, 3MRC Laboratory of Molecular Biology, Cambridge, UK age, mice that express ovalbumin only in the SI epithelium were Introduction: Interleukin 9 is a pleiotropic pro-inflammatory cyto- used. Only DCs isolated from SI-draining MLN, but not the colon- kine mainly produced by T cells, beside B cells and mast cells. draining MLN, were able to present SI derived antigen and cause Recently, a specialized subset of T cells has been identified, dedicated proliferation of na€ıve T cells. to produce IL-9 and therefore named Th9 cells. In these cells the To investigate DC migration from PPs to the MLN, PPs in Kaede IL-9 gene is regulated by transcription factors like PU.1 and IRF4. mice were photoconverted. DCs were then detected in the SI-drain- IL-9 has been found to exert many biological functions, but the role ing MLN, indicating that some DCs migrate from the PP to the of Th9 cells in colitis and colitis-associated cancer is still unclear. MLN. Migration of PP DCs to the MLN was reduced in CCR7-/- High concentrations of IL-9 in the colon tissue of colitis patients mice and in mice pre-treated with FTY720. Thus, we have identified point out the important role for this cytokine in the development of that a proportion of DCs migrate from the PP to the MLN by a colitis, but the involvement of Th9 cells in antitumor immunity is CCR7 and S1P dependent mechanism. These findings open new still not known. For this reason we started to investigate the role of avenues for controlling the functions of intestinal DCs, and there- IL-9 and Th9 cells in the development of colitis-associated colorectal fore manipulating the intestinal immune response, with exquisite cancer (CAC). precision. Methods: Il9KO mice were subjected to the experimental oxazolone model to induce colitis and treated with AOM/DSS for the development of colon cancer. Miniendoscopic analysis has been done to monitor the inflammation and the number and size of tumors. For 083 restoration of the IL-9 deficient phenotype an IL-9 expressing mini- The effect of oral Salmonella Typhimurium infection on circle DNA vector was injected into Il9KO mice by hydrodynamic intestinal mononuclear phagocytes injection and the model of AOM/DSS has been investigated. Tumors L. Utriainen1, V. Cerovic1, S.A. Houston1, D.M. Wall1, of the colon have been isolated for histological sections and stained M.J. Wick2 & S. W.F. Milling1 with T cell specific antibodies and PU.1. For analysis of cytokines 1Institute of Infection, Immunity and Inflammation, University of and transcription factor expression RT-PCR was performed. Glasgow, Glasgow, UK, 2Department of Microbiology and Immunology, Results: As Il9KO mice were protected in the experimental oxazo- University of Gothenburg, Gothenburg, Sweden lone-colitis model, we analysed those mice in the tumor model. We observed a significant reduction of tumor size and numbers in Il9KO Recent literature contains contradictory data regarding the functions, mice. Furthermore, we explored the effect of IL-9 on tumor growth and even the identification of the antigen presenting cells (APCs) in by overexpression of IL-9 via mini-circle DNA and AOM/DSS treat- the intestine that induce responses after infection with Salmonella ty- ment, which led to the development of tumors when IL-9 is absent. phimurium (STM). We have developed tools to unambiguously Additionally, in cryosections of tumorigenic colon tissue more PU.1+ identify intestinal CD11c+ APC populations, and have begun to T cells were found, pointing out the important relevance for Th9 examine the changes in dendritic cells (DCs) and macrophages that cells in colon cancer. occur after oral infection of antibiotic-treated mice with STM.

To investigate intestinal phagocytes, WT C57BL/6 or CX3CR1+/GFP not have a major effect on the biomarker profile or innate cell func- mice were treated with an antibiotic cocktail for 14 days, rested for tions in healthy active adults. 48 hours and infected orally with DinvG strain of STM. Mononu- clear phagocyte populations in the small intestinal lamina propria (LP) and mesenteric lymph nodes (MLNs) were analysed by flow cytometry 48 h after infection with STM. For lymph analysis, the 085 thoracic duct of mesenteric lymphadenectomised mice was cannu- The gut-associated lymphoid tissues in small intestine, not the lated 24 h after STM infection. Lymph was collected for 16 h, and large intestine, play a critical role in oral prion disease analysed by flow cytometry. pathogenesis No differences were observed in the frequency or number of total 1 2 1 hi D. Donaldson , K. Else & N. Mabbott migrating CD11c+ MHCII DCs in the MLNs after STM infection, 1 2 The Roslin Institute, Edinburgh, UK, University of Manchester, nor were there changes in the frequencies of DC subsets. However, Manchester, UK analysis of small intestinal LP phagocyte populations revealed small, but significant, differences in both CD64+ macrophages as well as in Many prion diseases, characterised by the accumulation of PrPSc, an DC subsets between infected and non-infected mice. Furthermore, abnormally folded isoform of the cellular prion protein (PrPC), are significantly higher proportions of CD11c+ MHCII+ DCs were pres- acquired orally. Following oral exposure, high levels of PrPSc accu- ent in lymph collected from infected animals, and within these there mulate upon PrPC-expressing follicular dendritic cells (FDC) in gut- was a significant increase of the CD103+ CD11b+ DC subset. Fur- associated lymphoid tissues (GALT) prior to neuroinvasion. Accu- thermore, no CX3CR1hi expressing cells were detected in lymph of mulation of PrPSc in large intestinal GALT has been used diagnosti- STM infected animals. cally in humans and other animal species; however, the role of In conclusion, we provide here a detailed analysis of the intestinal GALT in large intestines in oral prion disease pathogenesis was phagocyte populations after STM infection. Oral infection with STM unknown. In order to precisely distinguish between the influence of is reported to cause specific changes in the myeloid cells that migrate GALT in the small and large intestines in oral prion pathogenesis, from the intestine. We have now been able to conclusively show that we created mice that were specifically deficient in FDC-containing oral STM infection does indeed lead to an increase in the proportion GALT only in the small intestine. Oral prion disease susceptibility of migrating DCs from the intestine, and to changes in the frequen- was blocked in the specific absence of the GALT in small intestine. cies of migrating DC subsets. In addition, we demonstrate that Although these mice had FDC-containing GALT throughout their CX3CR1hi macrophages do not migrate into lymph after STM infec- large intestines, these tissues did not accumulate prions and the mice tion. were refractory to infection after oral exposure. We also studied whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris at the time of prion exposure did not signif- 084 icantly affect disease pathogenesis. Together, these data definitively The effect of probiotic consumption on routine haematological demonstrate that FDC-containing GALT in the small intestine are and immune markers in a cohort of healthy active adults the critical early sites of prion accumulation and neuroinvasion after oral exposure. In contrast, large intestinal GALT does not influence M.J. Lehtinen1, N.P. West2, P.L. Horn3, S. Barrett2, prion pathogenesis during steady-state or inflammatory conditions. H.S. Warren4, G. Koerbin5, M. Brun5, D.B. Pyne6, The demonstration that GALT in the small intestine, not the large S.J. Lahtinen1, P.A. Fricker3 & A.W. Cripps2 intestine, are the important sites of prion accumulation and neuroin- 1Nutrition and Health, Sokeritehtaantie 20, Kantvik, Finland, 2Griffith vasion has important implications for our understanding of the fac- University, Australia, 3Australian Institute of Sport, Australia, 4The tors influencing the risk to infection and the pre-clinical diagnosis of Canberra Hospital, Australia, 5ACT Pathology, Australia, 6James Cook disease. University, Australia In recent meta-analyses, a positive effect of probiotics against upper respiratory tract illnesses has been noted, but the underlying immune system changes are still mostly obscure. We have reported in a 086 three-arm clinical trial ((i) Bifidobacterium lactis Bl-04 (Bl-04), (ii) The inhibition of IKB kinase ameliorates experimental colitis in Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 rats (NCFM+Bi-07), or (iii) placebo) that daily consumption of Bl-04 by 1 1 1 1 A.R. Nogales , F. Algieri , J. Garrido-Mesa , P. Utrilla , healthy active adults reduces the risk of experiencing upper respira- 1 2 1 M. Rodrıguez-cabezas , C. Thiemermann &J.Galvez tory illness (URI) episodes during 5-month winter season. To study 1 2 Pharmacology, Granada, Spain, Nephrology and Critical Care, immune system changes, pre and post supplementation blood sam- London, UK ples were drawn from a cohort (n = 144) of the study subjects. The samples were analyzed for electrolytes, liver and thyroid function, Introduction: The transcription factor NFjB seems to play a crucial leukocyte counts, plasma immune biomarkers (n = 34), tumoricidal role in the pathogenesis of IBD conditions. In this study we have NK-cell activity, and phagocytic activity of granulocytes and mono- investigated the impact of IKK inhibition on TNBS experimental cytes. Differences in plasma calcium (P = 0.03) and urea (P = 0.015) colitis in rats. were observed between the probiotic and placebo groups, however Methods: Wistar rats (200 g) were assigned to five groups (n = 10): these were within assay-specific established laboratory reference ran- Non colitic, control colitic and colitic groups treated intraperitone- ges.The results showed that in Bl-04 group the level of plasma ally with IKK 16 (inhibitor of IKK) (0.3 and 1 mg/kg/day), and chemokine MIP-1d (CCL15) increased by 29 % compared to pla- dexamethasone (1.2 mg/kg/day), starting the same day of TNBS coli- cebo. In NCFM+Bi-07 group there was a 7.7 % increase in total tis induction (10 mg). Rats were sacrificed one week after, and colo- lymphocytes and 11 % increase in plasma matrix metalloproteinase nic damage was assessed histologically and biochemically, by 1 level. There were no changes in granulocyte, monocyte or NK-cell myeloperoxidase activity (MPO). In addition, colonic qPCR analysis activities between the study groups. In conclusion, probiotic supple- was performed to evaluate the expression of pro-inflammatory cyto- mentation with Bl-04 or NCFM+Bi-07 is considered safe and did kines (TNFa, IL-1b, IL-6, IL-12, IL-17), the enzyme iNOS, chemo-

kines (MCP-1 and CINC-1), the adhesion molecule ICAM-1, as well 088 as proteins involved in the preservation of intestine epithelia (muc- The liver: An underestimated effector site of the intestinal IgA ins MUC-2 and MUC-3, trefoil factor TFF-3 and villin). response? Results: IKK 16 administration resulted in an intestinal anti-inflam- L. Moro-Sibilot1, P. Blanc1, A. Sanlaville1, J. Gaillard1, matory effect as evidenced macroscopically by a reduced extension 1 1 2 1 of colonic damage in comparison with control colitic rats. Biochemi- M. Chevandier , E. Bardel , A. Traverse-Glehen , T. Defrance , 1 3 cally, a decrease in colonic MPO activity was observed, indicating a D. Kaiserlian & B. Dubois lower neutrophil infiltration in the inflamed tissue. qPCR evaluation 1International Center for Infectiology Research, 21, avenue Tony of cytokines expression showed that IKK 16 treatment decreased Garnier, Lyon, France, 2Pathological Anatomy and Cytology Unit, colonic expression of the proinflammatory cytokines TNFa, IL-1b, HCL, CHLS - 165 chemin du grand Revoyet, Pierre-Benite, France, IL-6, IL-12 and IL-17. In addition, the IKK inhibitor reduced the 3Tumor Cells and their Immune Environment, Cancer Research Center expression of the chemokines CINC-1 and MCP-1, the inducible Lyon, 28 rue Laennec, Lyon, France enzyme iNOS, and the adhesion molecule ICAM-1. In addition, this IgA, the most abundant immunoglobulin of the body, maintains compound significantly up-regulated the expression of markers of host-microbiota homeostasis and protection against infectious intestine epithelial integrity. These effects were similar to those microbes, without inducing overt tissue inflammation. IgA isotype obtained with dexamethasone. switching and B cell differentiation into plasma cells [PC] are mainly j Discussion and Conclusion: The inhibition of NF B signalling path- initiated in mucosal-associated lymphoid tissues [MALT] and way by the IKK inhibitor IKK 16 results in beneficial effects in TNBS involve a range of cells and factors involved in oral tolerance. Capi- rat colitis through an improvement of the exacerbated immune talizing on our recent demonstration that the liver is a critical induc- response that characterizes the colonic inflammatory process. tive site of T cell tolerance to intestinal antigens [Ag] (drained to the liver via the portal vein), and is enriched in tolerogenic dendritic cells which favor IgA responses, we explored the role of the liver in 087 the IgA response at steady state and after oral immunization. Mouse liver at steady state contains around 20% of B cells, The intestinal mucus phenotype in Clca3 deficient mice including a majority of IgD+ follicular B2 cells, few B1 cells, and is E. Nystro¨m, L. Arike, G. Hansson & M.E.V. Johansson enriched in IgA PC (75% of liver PC), compared to the bone mar- Medicinal biochemistry, Medicinaregatan 9A, G€oteborg, Sweden row, mesenteric LN, spleen and blood. In addition, we found that + The intestinal mucus has been shown to be important for innate human liver contains around 5% of CD19 cells, mostly represented immunity by keeping bacteria at a distance. The MUC2 protein is by PC, half of which producing IgA. These IgA PC are localized in the main component in the mucus, giving the mucus its features the hepatic parenchyma close to portal tracts. and network-like structure. Proteomics has also revealed Clca3 (chlo- Oral immunization with the T-cell-independent Ag NP-Ficoll and ride channel, calcium activated 3) as one of the most abundant non- cholera toxin as adjuvant generated specific IgA producing cells in mucin proteins found in the intestinal mucus. Clca3 was first pro- the liver and intestinal lamina propria, suggesting that the liver posed to be a calcium regulated chloride channel but is today con- might constitute an alternative site of the IgA response. Using a sidered to be fully secreted. Despite extensive research the function transfer model of NP-specific transgenic B cells from Quasi Mono- of Clca3 in the mucus is still obscure. clonal mice, we found that oral immunization with NP-Ficoll The domain architecture which includes a metallohydrolase induced IgA-expressing B cells initially in Peyer’s patches, and later + + domain followed by a von Willebrand factor type A domain suggests IgA CD19lowCD138 cells in the liver, indicating that IgA isotype both protease activity and potential binding to other proteins. The switching in MALT was rapidly followed by homing of IgA plasma- abundance of Clca3 in mucus together with the domain architecture blasts/PC to the liver. In this respect, a unique phenotype in term of has led to the hypothesis that Clca3 has an important and structural i) proliferative state, ii) expression of B cell and PC markers, and iii) role in the intestinal mucus. homing molecules (chemokine receptors CXCR-4, CCR-9, CCR-10, a b We have examined the mucus phenotype in Clca3 deficient mice integrin 4 7) was observed on liver IgA PC at homeostasis. Taken to test our hypothesis. MUC2 expression and distribution has been together, these data indicate that the liver constitutes an early site of investigated by immunohistochemistry which has also allowed exam- homing of IgA PC primed in the intestine. ination of the mucus structure. Further, the mucus layer thickness, We propose that both in mice and human, the liver may be an growth, tissue attachment and responsiveness to stimulation were underestimated effector site for the mucosal IgA response, which examined in an Ussing chamber-like ex vivo explant system. The may maintain local homeostasis and fuel the duodenum with IgA barrier function of the mucus against bacteria was assessed by an ex translocating via the bile. vivo penetrability assay in parallel with fluorescent in situ hybridiza- tion of Carnoy-fixed tissue sections. In addition, a proteomic comparison between wild-type and Clca3-/- mucus was performed to 089 look at differences in other major mucus components. The mucosal environment influences the systemic inflammation observed in Spondyloarthropathy patients P.B. Wright1, H.M. Wessel1, A. McEntegart2, D. McCarey2, D. Gaya2 & S. W.F. Milling1 1Institute of Infection, Immunity and Inflammation, University of Glasgow, UK, 2Department of Rheumatology, Glasgow Royal Infirmary, UK Ankylosing Spondylitis (AS) is a chronic systemic inflammatory disease belonging to the spondyloarthropathy (SpA) group of genetically and pathophysiologically related diseases. Alongside inflammation within the vertebral column and sacroiliac joints, AS patients often develop extra-articular disease manifestations, includ-

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 39 ing inflammatory bowel disease (IBD). Specifically, approximately parenteral immunization and were detectable for at least two weeks 10% of AS patients develop overt IBD whilst 60–70% of AS patients with at most 1.5 % KLH-specific Treg per CD4(+) T cells. At fol- show evidence of subclinical intestinal inflammation. Dendritic cells low-up after six months no KLH-specific Treg were found but these (DCs) activate na€ıve T cells and maintain the balance between reappeared quickly three days after reimmunization; similar kinetics immunity and tolerance, and may therefore contribute to controlling were found in the CD4(+) CD127(-), the CD4(+) CD137(+) CD154 inflammation in AS. To understand the contribution of DCs to AS, (-) and the CD4(+) CD45RA(-) Foxp3(+) populations. Ten days it is important to establish their role in driving systemic and tissue- after initial parenteral immunization with the neoantigen KLH anti- localised inflammatory responses in patients. We therefore character- gen-specific Treg can be detected in the peripheral blood. As ised circulating and intestinal DC populations, to elucidate the expected for a secondary immune response Treg reappear quickly unique and shared immunological pathways driving these intercon- after reimmunization at follow-up. The effect of oral KLH on anti- nected diseases. gen-specific Treg induction remains to be analyzed. Compared to age-matched HCs, blood from AS patients contained a significantly higher ratio of pro-inflammatory (CD16+ À CD14 CD11c+) mononuclear cells compared to CD1c+ DCs. This CD16+ population induced CCR6 expression on responding na€ıve T 091 cells and promoted secretion of the Th17-associated cytokines IL-1b The novel oral vaccine delivery technology SmPill significantly and IL-6. Interestingly, the frequency of circulating CCR9+ CD4+ T increases vaccine-specific intestinal antibody responses cells in AS patients increased with inflammation, indicating potential S. Longet1, C. Davitt1, E. McNeela1, V. Aversa2, J. Tobias3, mucosal involvement. We have now developed robust methods for M. Lebens3, M. Rosa2, J. Holmgren3, I. Coulter2 & E.C. Lavelle1 purification of live cells from fresh intestinal tissue, allowing enu- 1Trinity College Dublin, Dublin, Ireland, 2Dublin City University, meration and functional analyses of intestinal myeloid populations. 3 À À Dublin, Ireland, University of Gothenburg, Gothenburg, Sweden Initial analyses of intestinal DC subsets (CD45+ CD14 CD64 CD11c+ MHC II+ CD103+/-) in health and disease have been per- Enteric infections are a major worldwide health problem causing formed. high levels of mortality and morbidity especially in developing coun- In summary, we have identified a shift in circulating myeloid tries. Oral vaccination is regarded as the optimal means to fight populations towards the CCR6-inducing CD16+ population that may intestinal infections as it elicits immune responses at intestinal and contribute to the induction of T cell-mediated AS pathogenesis. Fur- systemic levels. However, one of challenges of oral vaccination is the thermore, our data implicate mucosal DCs in driving systemic degradation of active components by stomach acids and enzymes. inflammation in AS patients. Our analyses of live cells prepared from This challenge can be overcome by the use of a novel technology fresh intestinal tissue enable precise examination of the disease- named Single Multiple Pill (SmPill ). SmPill comprises 1-mm inducing tissue resident effector populations. We anticipate that microspheres allowing encapsulation of active components. A unique comparisons of these immune cell populations in AS and IBD coating surrounds the core of the microspheres protecting the pay- patients with healthy controls will reveal important details about load from destruction at low pH and allows its specific release at pathogenic mechanisms controlling disease development. higher pH in the small intestine. A formalin-killed whole cell entero- toxigenic Escherichia coli (ETEC) candidate vaccine and the effective oral adjuvant a-Galactosylceramide have been enclosed inside the microspheres. Microscopy studies clearly demonstrated aggregates of 090 bacteria inside the microspheres. In addition, simulated intestinal The neoantigen KLH induces antigen-specific regulatory T cells fluid (pH 6.8) was shown to induce the disorganization of the inter- in humans nal structure of microspheres as well as the release of bacteria. Finally, we assessed that SmPill significantly increased the ability of 1 1 1 1 € 2 1 B. Boortz , T. Meyer , J. Maul , J. Preiß ,T.Dorner & R. Ullrich the ETEC vaccine to rapidly generate high titres of vaccine-specific 1Gastroenterologie, Hindenburgdamm 30, Berlin, Germany, 2 intestinal IgA and IgG in mice. Intestinal T and dendritic cell Rheumatologie responses as well as the underlying mechanisms of intestinal B cell Oral tolerance can prevent and treat autoimmunity in animal mod- responses leading to enhanced antibody responses are currently being els, but its efficacy in humans is unclear. We use the neoantigen key- analysed. In summary, these data highlight the potential that hole limpet hemocyanin (KLH) to healthy volunteers to investigate SmPill technology has to enhance the effectiveness of oral vaccine antigen-specific tolerance. Foxp3(+) CD4(+) CD25(+) regulatory T delivery. cells (Treg) are known for their suppressive function. Here we report our investigations about the systemic Treg population. Two groups of eight healthy volunteers each were compared. One group received 092 50 mg KLH per day orally for ten days, the other did not and served as control. Both groups were then subcutaneously immunized with The phagocyte oxidase in infection: A gut perspective KLH and a DTH skin reaction was provoked by an intradermal G. Pircalabioru1, M. Kubica1, G. Aviello2, B. Bourke2 & KLH injection. Follow-up investigations were done six months later. U.G. Knaus1 At selected time points, PBMC were isolated and samples were stim- 1Conway Institute, University College Dublin, Belfield, Dublin 4, ulated for 16 hours by anti-CD28, by anti-CD28 in combination Dublin, Ireland, 2National Children’s Research Centre, Our Lady’s with KLH, and by staphylococcal enterotoxin B (SEB). Afterwards Children’s Hospital, Crumlin, Dublin, Ireland the samples were stained for CD4, Foxp3, CD25, CD127, CD45RA, CD137, CD154, CD14, CD19, and analyzed by flow cytometry. Well recognized for their role in phagocyte-mediated pathogen kill- The study is ongoing and three subjects were analyzed so far. ing, reactive oxygen species (ROS) such as O2 and H2O2 are also an Comparing cells stimulated by anti-CD28 combined with KLH and integral part of the host defense system and inflammatory responses anti-CD28 alone, KLH-specific Treg were identified as the Foxp3(+) at mucosal surfaces. The cellular sources of pathogen-triggered ROS CD4(+) CD25(+) population after the exclusion of CD14(+) CD19 generation are predominantly NADPH oxidases. The best character- (+) L/D aqua(+) cells. Treg were seen only after parenteral immuni- ized member of the NADPH oxidase family is the phagocyte zation but not after oral antigen alone. Treg appeared ten days after NADPH oxidase (Nox2 complex). Its critical role in host defense is

highlighted by the life-threatening recurrent fungal and bacterial 094 infections observed in patients with chronic granulomatous disease The role of follicular dendritic cells in CTA1-DD mediated (CGD), a genetic disorder in which Nox2 function is defective due germinal centre formation to mutations in its essential subunits. We used Nox2 knockout (KO) S. Schussek, J. Mattsson, K. Schon€ & N. Lycke mice for investigating the interplay between inflammation, anti-bac- Biomedicine, Gothenburg, Sweden terial immunity and microbiota afterCitrobacter rodentium infection. Infection with upregulated expression of Nox1 and Duox2, the main In the germinal centre (GC), follicular dendritic cells (FDC) provide oxidases expressed in the intestinal barrier, in a similar manner in a depot of antigen and support the survival of antigen-specific B both wild type (WT) and KO mice. However, loss of Nox2 activity cells, which can then be induced to differentiate into high-affinity B and consequently of ROS generation increased the overall pathology cell clones, long-lived plasma cells or memory B cells by follicular T after oral challenge with C. rodentium. Nox2-deficient mice showed helper cells (Tfh). Specific targeting of adjuvants and vaccines to cell increased pathogen colonization followed by pronounced mucosal populations critical for the induction and augmentation of GC and submucosal colon damage. Changes in the microbiota composi- responses could greatly improve vaccine safety and enhance vaccine tion affect the physiology and homeostasis of the host GI tract. efficacy. A derivative of cholera toxin, the CTA1-DD adjuvant was Although C. rodentium infection changed the microbiota complexity exceptionally effective at promoting the formation of large and in WT and KO mice, the shift was more pronounced in Nox2 KO numerous GC, as well as enhanced antibody responses and memory mice with a significant increase of the Bacteroidetesphyla. Loss of B cells development. We previously explored the in vivo distribution Nox2-derived ROS caused overgrowth of Enterobacteriaceae, a family of CTA1-DD in tissues and observed that CTA1-DD- mediated acti- of bacteria previously linked to host gut inflammation. Long term vation of complement and binding to follicular dendritic cells via antibiotic treatment induces dysbiosis and susceptibility to infection complement receptor 2 was crucial for adjuvant-induced GC forma- and colitis. Our results reveal that dysbiotic Nox2 KO mice infected tion and antibody production. On-going analysis of gene-expression with C. rodentium exhibited poor survival despite enhanced Nox1 levels in FDCs following CTA1-DD immunisation suggests a direct and Duox2 levels. Even though pathogen colonization of the GI tract effect of the adjuvant on FDC functionality and their capacity to was similar to dysbiotic WT mice, Nox2 KO mice showed bacterial promote GC formation. In order to isolate and track FDCs more translocation to liver, spleen and cardiac blood. These findings sug- easily, we have created a mouse model expressing the GFP marker gest a role of phagocyte-derived ROS in limiting bacterial dissemina- gene under the CD21 promotor. Using this model, we are assessing tion and systemic disease in the absence of microbiota-mediated the activation status and localisation of FDCs and Tfh in response to protection. CTA1-DD, which will delineate the key elements required for the adjuvant’s effects and potentially identify key regulators of GC B cell activation, memory development and vaccine-induced protection.

093 The role of dendritic cells in inducing Th2 responses to schistosoma mansoni eggs 096 The viability of lactobacillus fermentum CECT5716 is not J. Mayer1, L. Webb2, S. Houston1, V. Cerovic1, A. MacDonald2 & required to exert its intestinal anti-inflammatory activity S. Milling1 1Institute of Infection, Immunity and Inflammation, 120 University T. Vezza1, A. Rodrıguez1, F. Algieri1, N. Garrido1, A. Zarzuelo1, Place, Glasgow, UK, 2MCCIR University of Manchester, 46 Grafton M. Olivares2, M. Comalada3, M.E. Rodrıguez1 &J.Galvez1 Street, Manchester 1Pharmacology, Granada, Spain, 2Research Department of Biosearch, Granada, Spain, 3Institute for Research in Biomedicine, Barcelona, Schistosoma mansoni eggs (SME), which are the main cause of Spain chronic Schistosomiasis, provoke a strong Th2 immune response that can lead to granulomatous reactions and fibrosis in affected Introduction: Lactobacillus fermentum CECT5716 is a probiotic organs. Recently, CD11c depletion has been shown to severely dis- with immunomodulatory properties that has been reported to show rupt Th2 immune responses against Schistosoma mansoni, suggest- intestine anti-inflammatory activity in experimental colitis. Some ing that antigen presenting cells, which express CD11c, mediate this studies have proposed that the viability of the probiotics is not immune response. Dendritic cells (DCs) are the most likely CD11c- essential to exert their beneficial effects. In the present study, we positive candidates, as they continuously migrate from epithelia, car- studied if the administration of viable Lactobacillus fermentum is rying antigens, to the draining lymph nodes where they activate necessary to obtain its intestine anti-inflammatory effect. Also, in vi- naive T-cells. However, the precise roles of DCs in inducing immune tro studies were performed to confirm these observations. responses against SME have not been studied. Methods: Lactobacillus fermentum CECT5716 was administered We have developed an in vivo mouse model to inject SME in the orally (5 x 108 CFU/rat/day) to Wistar rats, live or dead (95C for subserosal layer of the small intestine of C57BL/6 mice, the anatomi- 30 min) for two weeks before colitis induction, and thereafter until cal location where SME naturally become lodged. This provokes a colonic evaluation one week later, both macroscopically and bio- Th2 response in the draining mesenteric lymph nodes (MLNs). To chemically: myeloperoxidase activity, glutathione content, TNFa and test the role of DCs in this response, we will purify migrating DCs IL1b levels, as well as iNOS expression. In vitro experiments were from egg challenged mice and control animals and then transfer performed in Caco-2 (intestine epithelium) and RAW 264.7 (macro- these cells into the subcapsular region of the MLN of naive mice. phages) cells, which were incubated with the probiotic (live or Detection of an antigen-specific Th2 response in those mice will death) (108 CFU/ml), and stimulated with LPS (100 ng/ml) or IL- indicate that migratory DCs are sufficient to carry and present egg 1b (1 ng/ml) for 30 minutes (western blot) or 24 h (IL-8 or nitrite antigens and drive Th2 responses in our model. determination). This knowledge will contribute to a better understanding of how Results: The probiotic, live or dead, showed intestine anti-inflamma- the early immune response against SME is mediated. This informa- tory effect, by reducing the colonic damage area. Biochemically, tion can be applied to battle chronic Schistosomiasis and may help reduction in MPO activity, indicating lower neutrophil infiltration, understand Th2 driven responses against other parasites or Th2 and increase in glutathione content was observed in treated groups. allergic responses. Also, both treatments reduced the levels of the proinflammatory

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 1), 12–42 Abstracts 41 cytokines TNFa and IL1b, and inhibited the increased iNOS expres- response of macrophages to internalized bacteria. Cell surface TLR2 sion in colitic rats. In vitro studies showed that live or dead probiot- initiated an inflammatory response while subcellular TLR2 negatively ic inhibited the stimulated production of IL-8 (Caco-2 cells) and regulated a TLR4-mediated hyper-inflammatory response that was nitric oxide (RAW 264.7) in a similar way. In the epithelial cells, this phagocytosis-dependent and driven by a type I interferon autocrine inhibitory effect was associated with a reduced phosphorylation of loop. TLR2-deficient macrophages were hyper-responsive to bacteria p42/44 ERK and SAPK/JNK when compared with stimulated cells and developed an M1-like phenotype, which was absent in TLR2/ without probiotic. TLR4-deficient macrophages. Our findings identify subcellular TLR2 Discussion and Conclusion: The viability of L. fermentum as being important for the negative regulation of inappropriate CECT5716 to downregulate the stimulated immune response and TLR4-mediated inflammatory responses to bacteria. exert its intestine anti-inflammatory effect is not essential.

099 097 TRPV1 expression in spleenocytes of different mouse strains TLO Developmental Program: Divergent paradigm of lymphoid could be critical in understanding variations in immune organogenesis responses S. Nayar1, J. Campos1, B. Glaysher2, J.H. Caamano1, M. Bakri1, M. Jaleel2 & P. Joanne3 D.R. Withers1, K.M. Toellner1, S. Luther3, T.D. Randall4, 1King Abul Aziz University, Saudi Arabia, 2University of Manchester, M. Coles2, C.D. Buckley1 & F. Barone1 United Kingdom, 3Univeristy of Manchester, United Kingdom 1Immunity and Infection, University of Birmingham, Birmingham UK, Objectives: This study discusses for the first time TRPV1 expression, 2Centre of Immunity and Infection, University of York, UK, functionality and subsequent release of CGRP from immune cells in 3Department of Biochemistry, University of Lausanne, Switzerland, the spleens of AKR, BALB/c and C57. 4Division of Clinical Immunology, UAB, USA Material and Methods: TRPV1 expression on spleenocytes: Spleen Tertiary lymphoid organs (TLOs) represent the histological hallmark cells were disassociated and stained with different dilutions of 6 of many immune-mediated inflammatory diseases. TLOs are charac- flourochromes. conjugated antibodies for CD11c, F480, CD4, CD8, terized by a functional leukocyte aggregation and network of lym- CD19, IgM, IgD, Gr1 were then along with TRPV1 biotinylated anti- phoid-like stromal cells (LLSc). LLSc express lymphoid chemokines body were administered. Calcium signaling: Indo-1 was administered (CXCL13, CCL19, and CCL21), survival factors (BAFF and IL-7), to spleenocytes at 5x105 cells/sample. samples were treated with lymphoid markers and adhesion molecules (gp38, RANKL, ICAM-1 either capsaicin, anandamide or both at concentrations 6, 12, 25, 50, and VCAM-1) that locally support lymphocytes survival and organi- 100uM. specific cell types’ calcium signaling was examined with a zation in ectopic sites areas. Using a combination of inducible mod- single concentration of agonists (100uM). CGRP release from els of TLO formation we demonstrated that the acquisition of this spleenocytes: 1x106 cells per sample were treated with 100uM with lymphoid phenotype by the non-activated resident stroma requires a capsaicin, anadamide or both. The supernatants were collected multistep process, fundamentally different from that responsible of 5 minutes post treatment. The remaining cells were collected and secondary lymphoid organ formation. We showed that early, during sonicated. both supernatants and sonicated cells were examined for TLO formation, IL-4Ra engagement via IL-13 on quiescent tissue- CGRP via ELISA. resident fibroblasts induces LLSc priming and mediates the up-regu- Results: lation of gp38 and lymphoid-associated adhesion molecules. Expan- 1-TRPV1 is expressed on immune cells in spleen of all mouse sion of this activated lymphoid stroma requires IL-22/IL-22R strains in study (AKR, BALB/c and C57) mediated signaling. Lack of IL-22 or its receptor induces defective 2-BALB/c have a higher percentage of TRPV1 on CD11c+ cells, LLS proliferation, abrogation of CXCL13 expression and TLO invo- F4/80+ cells and CD11c+F4/80+ cells lution. Finally we demonstrated that, similarly to secondary lym- 3-AKR had higher percentage of TRPV1 on IgM+ cells, IgD+ cells phoid organ, stabilization of the stroma to a functional lymphoid and IgD+IgM+ cells. phenotype requires lymphocyte infiltration and lymphotoxin beta 4-C57 had significantly higher percentage of TRPV1 in CD8+ expression. This work highlights critical differences between the cells. embryonic program responsible for SLO formation and the inflam- 5-CGRP release appears to be impaired in AKR compared to matory development of TLOs and unveils novel potential targets for BALB/c post capsaicin treatment. TLO targeting. 6-CGRP inhibited Th1 cytokines in all three strains while promoting Th2. Conclusion: This study shows for the first time TRPV1 expression on immune cells in the spleen. Also, it discusses TRPV1 signalling 098 and subsequent release of CGRP. Findings are then studied in the TLR2 located in subcellular compartments negatively regulates light of the known immune phenotypes that all three mouse strains TLR4-mediated inflammatory response of macrophages to develop in reponse to the helminth trichuris muris (t.muris). This internalized bacteria study suggests that lack CGRP release in AKR is impaired compared L. Butto1, S. Melgar1, J. O’Callaghan2, I. Jeffery1, H. Harris2, to BALB/c and C57 mice. J. MacSharry1, F. Shanahan1, P. O’Toole1 & K. Nally1 The Authors would like to Thank KAU for their support of Dr 1Alimentary Pharmabiotic Centre, Cork city, Ireland, 2University Bakri M Assas. College Cork, Cork city, Ireland Recognition of microbes by toll-like receptors (TLRs) is critical for initiation of appropriate innate and adaptive immune responses. How cross-talk between TLRs situated in various cellular locations contributes to host-microbe dialogue and immunoregulation is pres- ently unclear. Here we report a dual role for TLR2 in regulating the

101 vivo. Tracer molecule binding was analyzed using fluorescence Vagus nerve modulates colonic inflammation in an alpha-7 microscopy. nicotinic receptor (a7AChR) independent mechanism Results: When administered from the apical side, only dextrans that passed the epithelial barrier bind to the scavenger and hence label M.D. Giovangiulio, N. Stakenborg, G. Bosmans, G. Farro, sites of increased permeability to macromolecules at a resolution in P.J. Gomez-Pinilla, G. Matteoli & G.E. Boeckxstaens the µm-range. Using standard epithelial two-compartment culture Department Clinical and Experimental Medicine, KU Leuven, Leuven, systems spontaneous macromolecule passage was detected in all cell Belgium lines analyzed. Passage was generally rare, occurred through big leaks To preserve intestinal immune homeostasis, our immune system has very inhomogeneous in distribution and magnitude and was reduced evolved redundant regulatory strategies to prevent inappropriate with increased culture duration. The basolateral label increased when immune reactions against innocent luminal antigens. Recently, we barrier function was disrupted with EGTA or TNFalpha, and corre- have shown that the parasympathetic nervous system through the lated well with a corresponding global flux measurement. vagus nerve (VN) dampens intestinal muscular macrophages via Conclusion: We propose a new imaging approach that allows for alpha-7 nicotinic receptor (a7AChR) in a model of postoperative local analysis of macromolecule passage which provides new insights ileus. Similarly, we showed that vagotomy induces loss of oral toler- in immunologically relevant leaks in epithelial barriers in health and ance by reducing the conversion of antigen-specific Tregs in the lam- disease. ina propria and increases the severity of colitis. However, the role of a7nAChR and the immune cells involved in the vagal control of colitis is still not clearly defined. To study the influence of a7nAChR on Th cells, two groups of RAG1-/- mice were injected respectively 103 with na€ıve Th cells from WT or a7nAChR-/- mice. To define the Vitamin-D status and its regulation of cd T-cells in Behcßet’s role of a7nAChR on innate immune cells, a7nAChR-RAG1 double disease € knockout mice were injected with WT naıve Th cells. In both experi- M.D. Samiul Hasan, L.A. Bergmeier & F. Fortune mental approaches, recipient mice developed similar severity of coli- Institute of Dentistry, Queen Mary University, 4, Newark street, tis as shown by histology score, cytokine expression and colonic Th London, UK cells polarization. Altogether, our results suggest that a7nAChR deficiency either on Th or on innate immune cells is not crucial for con- Background: Behcßet’s disease (BD) is a multisystem inflammatory trolling colonic inflammation. Further, studies will be required to disorder characterized by oro-genital ulcerations, ocular manifesta- define if other nicotinic receptors or other neurotransmitters are tions, arthritis, vasculitis etc. with episodes of exacerbations and responsible for the vagal anti-inflammatory effect in the colon. remissions. BD is often regarded as at the crossroad between autoimmunity and auto-inflammation however, the pathogenesis remains inconclusive. Besides genetic predisposition, immune-dysregulation involving cytotoxic lymphocytes such as cd T-cells is reported to 102 have a role. Vc9Vd2, a major subset of these atypical T-cells are Visualizing macromolecule passage through epithelia present in the inflammatory lesion and lead to rapid cytokine production, particularly of Th1-type when exposed to bacterial phosp- J.F. Richter1, R. Schmauder2, A. Gebert1 & M. Schumann3 hoantigens and thus might be responsible for inducing and/or 1Department of Anatomy II, University Hospital Jena, Jena, Germany, maintaining the pro-inflammatory environment characteristic of the 2Department of Physiology II, University Hospital Jena, Kollegiengasse disease. Vitamin-D (Vit-D) has regulatory properties for immune 9, Jena, Germany, 3Department of Gastroenterology, Charite University system function and has been implicated as a protective factor in Medi, Hindenburgdamm 30, Berlin, Germany some diseases but to date, no studies have addressed the effect of Introduction: Epithelial layers are the body´s interfaces between Vit-D on cd T-cells in BD. internal and external compartments. As physical barriers to poten- Materials and Methods: Serum 25-hydroxyvitamin-D levels of BD tially antigenic macromolecules epithelia prevent uncontrolled access patients were measured. Vc9Vd2 T-cells from BD patients were pre- to immune competent compartments. The current discussion about treated with Vit-D, followed by stimulation with the phosphoanti- disease initiation in IBD focusses on mucosal barrier function, since gen, HDMAPP. Antigen induction of IFNc, TNFa and IL10 expres- loss of barrier function appears to trigger an untempered immune sion was measured using flow cytometry and ELISA. reaction to the commensal intestinal flora. Progress in understanding Results: 80% of BD patients are deficient in serum 25-hydroxyvita- such barrier defects is largely hampered by a lack of methods that min-D levels. In-vitro treatment with Vit-D significantly reduced can locally resolve passage sites of macromolecules. To date, epithe- IFNc (P = 0.002) and TNFa (P = 0.001) expression by Vc9Vd2T- lial macromolecule passage is analyzed using global measurements cells. There was no effect on IL10 production. such as tracer flux studies which average contributions from many Discussion and Conclusion: The data indicate a role of Vit-D cells. As these global measurements do not locally resolve barrier in Vc9Vd2 T-cell regulation in BD. BD patients are deficient in defects, leak property distribution cannot be analyzed and rare Vit-D suggesting that this may contribute to the prolonged inflam- events may be missed. Therefore, local identification of barrier mation. Vc9Vd2 T-cell is a poor source of IL10 and there is breaks is interesting and needed. IL10 polymorphism in some BD patients which might explain the Aims: To localize and characterize macromolecule passage sites in IL10 data. Our data support the notion that Vit-D negatively regu- epithelia. late the proinflammatory function of cd T-cells and may be implied Methods: Using MDCKII, Caco-2/BBe, HT-29/B6 epithelia and rat as a protection against autoinflammatory and autoimmune disorders colonic mucosae we applied fluorescent dextran tracers of various including BD. sizes which bind to cell-adherent scavenger molecules at the basolateral membranes of single-layered epithelial sheets in vitro and ex